Homoharringtonine exhibits senotherapeutic activity that mitigates diet- and age-associated obesity and insulin resistance and extends lifespan in mice

Domain: neuroscience
Published: 2026-03-31
Editorial outcome: minor_revision

Abstract
The accumulation of senescent cells in white adipose tissue (WAT) is closely associated with the functional decline of WAT and plays a causal role in the pathogenesis of metabolic diseases. Therefore, the elimination of senescent cells in WAT holds promise for the treatment and prevention of age-related metabolic diseases. Using a drug-repositioning strategy for 2150 clinically applied compounds, we discover that homoharringtonine (HHT), an FDA-approved anti-leukemic drug, manifests senotherapeutic activity in vitro in multiple cell types including human preadipocytes, while inflicting minimal cytotoxicity to non-senescent cells. HHT treatment prevents diet- or age-induced metabolic abnormalities in male mice targeting senescent adipocytes and preadipocytes to improve WAT function and reduce WAT inflammation. Moreover, HHT treatment attenuates age-associated phenotypes of human adipose tissue. Mechanistically, the senotherapeutic effects of HHT are mediated through the direct interaction of HHT with heat shock protein family A member 5 (HSPA5). Importantly, we found that HHT treatment delays aging and extends the lifespan in progeroid and aged mice. Our study demonstrates the novel senotherapeutic potential of HHT to mitigate age- and obesity-related metabolic dysfunction and extend longevity in mice.

Introduction
Cellular senescence refers to a terminal state of growth arrest in which cells cease to divide, undergo morphological changes (enlarged and flattened shape), take on a senescence-associated secretory phenotype (SASP), and exhibit increased senescence-associated beta-galactosidase (SAβG) activity and expression of p53 and/or p161,2. Senescent cells release inflammatory cytokines, growth factors, and matrix metalloproteinases, resulting in the dysfunction of neighboring cells3,4. Consequently, the accumulation of senescent cells induces tissue aging and organ dysfunction, thereby contributing to the development of age-related chronic diseases. Therefore, the elimination of senescent cells or the blocking of the SASP extends the healthspan and prevents the emergence of age-related pathologies5–10.

The white adipose tissue (WAT) plays essential roles in whole-body metabolism, not only by storing excess energy and releasing lipids in response to energy deficits, but also by producing various adipokines and endocrine hormones. These metabolic functions of WAT require functional adaptation to the metabolic environment through the dynamic remodeling of adipose cellular composition. The remodeling capacity of WAT drastically declines with aging, with inadequate angiogenesis resulting in tissue hypoxia, chronic inflammation, and the deposition of excess extracellular matrix (ECM), ultimately leading to adipose tissue fibrosis. Adipose tissue fibrosis is a critical feature of senescence, which drastically increases with age11,12 contributing to further adipocyte dysfunction. Adipose tissue fibrosis thereby accelerates the progression of metabolic diseases, such as fatty liver disease and type 2 diabetes mellitus11,13. Currently, the therapeutic options for blocking or reverting age-related adipose tissue fibrosis and dysfunction are limited. Therefore, there is a need for pharmacological treatments that can halt or reverse the aging process to mitigate adipose tissue fibrosis and its related metabolic complications.

Interestingly, recent evidence suggests that the accumulation of senescent cells in WAT is a critical process in the pathogenesis of age-associated adipose tissue fibrosis and dysfunction, as it contributes to various metabolic aberrations. Young or non-senescent (NS) adipocyte precursor cells (APCs) in WAT can give rise to mature adipocytes and brown fat-like cells (beige adipocytes) in response to high energy intake and cold exposure, respectively14. However, senescent APCs in aged or obese WAT lose the capacity to differentiate into mature white adipocytes (i.e., adipogenesis) or beige adipocytes; rather, the APCs exhibit increased fibro-inflammatory properties, and the adipose tissue expands through increased size of adipocytes (i.e., hypertrophy). Senescent APCs display substantially increased expression of CD9, an age-associated APC marker, and exhibit limited differentiation capacity, leading to significantly reduced hyperplastic adipogenesis14,15. Additionally, the age-associated functional decline in mature adipocytes and adipose immune cells plays key roles in the pathological remodeling of adipose tissue16–18. Notably, eliminating senescent cells by senolytics, such as dasatinib with quercetin (hereafter D+Q), or activating drug-inducible suicide genes driven by the p16 promoter, alleviates high-fat diet (HF)-induced insulin resistance in animals19,20. Moreover, in a recent clinical trial, a single three-day oral administration of D+Q in diabetic patients with chronic kidney disease decreased senescent cell accumulation and inflammation in the adipose tissue21.

In this study, we tested 2150 clinically applied or FDA-approved compounds22 to find new senotherapeutics for the treatment of age- and obesity-related metabolic diseases. We discovered that homoharringtonine (HHT), a natural plant alkaloid approved by the FDA for the treatment of chronic myeloid leukemia (CML), manifests a senotherapeutic activity in cultured APCs. Additionally, HHT treatment attenuates the age-associated phenotypes in adipose tissue of diet-induced obese mice and individuals with obesity. Furthermore, our data demonstrated that HHT treatment mitigates the aging phenotype and extends the lifespan of both progeroid and naturally aged mice. Collectively, our study suggests that HHT is a promising senotherapeutic agent for addressing age-related diseases and promoting healthy aging.

Results
Drug repositioning approach to discover novel senotherapeutics
We screened a library comprising 2150 clinically applied compounds to identify novel senotherapeutics among existing drugs22. For the primary screening, we used doxorubicin-induced prematurely senescent (PS) human dermal fibroblasts (HDFs)23 in media containing 10% fetal bovine serum (FBS). Senescence phenotypes of PS HDFs were confirmed by SAβG staining and by assessing the protein expression levels of p21 and p16 (Fig. 1A, S1A, and S1B). PSHDFs were incubated with each of the compounds at 100 nM for 4 days, then cell survival and cellular senescence were assessed using the cytotoxicity assay and SAβG staining, respectively. The primary screening identified 110 compounds that increased cytotoxicity, or decreased SAβG staining in PSHDFs, which were thereafter regarded as senolytic, or senomorphic candidates, respectively (Fig. 1A).

To further verify the senolytic or senomorphic effects of the 110 compounds, we conducted secondary screening by testing these compounds in multiple cell types, including replicatively senescent (RS) HDFs, RS human umbilical vein endothelial cells (RSHUVECs), and PS human retinal pigment epithelial cells (PShRPEs) (Figs. S1C-H). Through the secondary screening of the 110 compounds, we selected 15 compounds that showed senolytic or senomorphic (hereafter senotherapeutic) activities across all three cell types (Fig. 1A, S1I, and Supplementary Table 1). ABT263 and rapamycin were used as senolytic24 and senomorphic25 positive controls, respectively (Fig. S1I).

Identification of HHT as a novel senotherapeutic molecule
Adipose tissue senescence plays a causal role in the development of obesity- and age-associated insulin resistance and metabolic dysfunction6,19. To validate the effects of the candidate compounds on obesity and associated metabolic dysfunction, we performed pilot in vivo experiments in high-fat diet (HF)-induced obese mice. Among the 15 candidates, two were initially excluded due to known adverse effects. We excluded #1806 (trametinib) due to gastrointestinal symptoms and reduced food intake26–28, and #231 (everolimus) due to hyperglycemia29,30. We tested the remaining 13 compounds in the HF-induced obese mice (Fig. 1B and Supplementary Table 2). We found that the #34 compound (HHT) mitigated weight gain and improved glucose regulation compared to PBS control in the absence of food intake differences (Fig. 1C–G and S2A–E). We then performed in vitro analyses to investigate cellular mechanisms underlying this effect in media containing 10% FBS. We found that HHT induced cytotoxicity in RS human visceral preadipocytes (HPAs) and RS HDFs in a dose-dependent manner, and elevated LDH levels in the media of these senescent cells, while reducing senescence-related phenotypes (Figs. S3A–C and S3E–G). This was accompanied by the elevation of cleaved caspase-3 (Casp-3) protein, indicating the induction of apoptotic cell death by HHT treatment in RS HPAs (Fig. S3D) and RS HDFs (Fig. S3H). Interestingly, HHT appeared to reduce cell numbers at 50 nM and 100 nM concentrations in non-senescent (NS) cells (Figs. S3A, S3B, S3E and S3F). Importantly, this did not induce LDH release (Figs. S3C and S3G), suggesting growth arrest rather than cell death, which aligns with the known mechanism of HHT in cancer cells and normal cells31,32. This finding was further supported by flow cytometry, which demonstrated increased G0/G1 arrest in HHT-treated NS cells (Fig. S3I). Given that HHT induces cell cycle arrest, it was necessary to clarify whether the observed decrease in NS cell numbers was due to actual cytotoxicity or simply growth inhibition. To address this, we repeated the experiments under 0% FBS conditions, where overall cell proliferation is minimized. Under these conditions, HHT induced significant cytotoxicity in senescent HPAs and HDFs while having no effect on NS cell numbers (Fig. 1H–K and S3J–M). These effects of HHT are specific to senescent cells and comparable to well-known senolytics, dasatinib + quercetin (D+Q) and ABT263 (Figs. S3N, S4, and Supplementary Table 3). Additionally, HHT treatment reduced the expression of some SASP proteins in all tested cell types, including HPAs (Fig. S5). These results suggest that HHT mitigates glucose intolerance in mice, and exhibits senotherapeutic activity across multiple human cell types, including preadipocytes.

HHT reduces adiposity and improves insulin resistance in HF-fed obese mice
To further investigate the beneficial metabolic effects of HHT on HF-induced obesity and metabolic dysfunction, we fed mice a 60% HF diet for 6 weeks, then treated with either HHT or PBS control three times weekly for 8 weeks while continuing the HF diet (Fig. 2A). Consistent with our in vivo screening studies (Fig. 1C), HHT treatment significantly reduced body weight gain from 4 weeks after the start of HHT treatment without a significant difference in accumulated food intake between the two groups (Fig. 2A, B), indicating that reduced body weight gain by HHT is not primarily attributed to decreased food intake. Importantly, we found a significant reduction in the fat weight in HHT-treated mice without significant effects on other organs (Fig. 2C and S6A), suggesting that reduced adiposity is the main contributor to body weight reduction. However, this effect was not observed in young mice fed a standard chow diet (Fig. S6B). Fasting plasma glucose levels were reduced by HHT (Fig. 2D) without a significant difference in fasting plasma insulin levels between the groups (Fig. 2E). Additionally, HOMA-IR was significantly lower in the HHT-treated mice than in PBS-treated mice with similar HOMA-%β level in both groups, suggesting that HHT improves HF-induced insulin resistance (Fig. 2F, G). To examine the effect of HHT on glucose homeostasis, we performed an intraperitoneal glucose tolerance test (IPGTT). The HHT-treated group showed enhanced blood glucose clearance compared to the PBS-treated group (Fig. 2H). The area under the curve (AUC) for glucose clearance was significantly lower in the HHT-treated mice compared with the PBS-treated mice (Fig. 2I). The plasma insulin levels and AUC for insulin were similar between the HHT- and PBS-treated mice (Fig. 2J, K). These results suggest that HHT reduces fat mass and improves insulin resistance in HF-fed obese mice.

HHT reduces cellular senescence and inflammation in WAT of HF-fed obese mice
To determine whether HHT improves glucose homeostasis in HF-induced obese mice by targeting adipose tissue senescence, we evaluated senescence markers in HHT-treated white adipose tissues (WATs). HHT treatment decreased SAβG staining in various adipose depots, including subcutaneous, epididymal, mesenteric, and perirenal fat tissues, to levels similar to those in the normal chow diet control (Ch-PBS) group (Fig. 3A). Indeed, histological analysis revealed that the SAβG-positive cells in the epididymal fat tissues were significantly increased by HF, while HHT treatment significantly reduced their abundance (Fig. 3B). To further confirm the senotherapeutic activity of HHT in vivo, we employed a transgenic p16-luc mouse model that carries the entire human p16 gene locus tagged with firefly luciferase and is reported to increase bioluminescence concurrent with the p16 level with age33. The p16-luc mice were fed with HF for 6 weeks and then treated with HHT or PBS three times a week for 8 weeks. Notably, bioluminescence was increased in the HF (HF-PBS) compared to the Ch-PBS group, and this elevation was normalized by HHT treatment (HF-HHT, Fig. 3C). These results indicate that senescent cells accumulated in the adipose tissues of HF-fed p16-luc mice, but were cleared by HHT, demonstrating the in vivo senotherapeutic activity of HHT. Furthermore, HHT treatment significantly reduced the expression levels of p53, p21, and p16 proteins in WAT when compared with those in HF-PBS (Fig. 3D). Collectively, these data suggest that HHT attenuated cellular senescence in the adipose tissues of the HF-fed obese mice.

Senescent cells accumulate in obese WAT and stimulate the secretion of inflammatory cytokines, immune modulators, growth factors, and proteases (i.e., SASP), leading to adipose tissue dysfunction and metabolic disturbance34. Notably, we found that HHT treatment reduced crown-like structures (asterisks in Fig. 3E, F), suggesting the anti-inflammatory effects of HHT in WAT. HF induces hypertrophic enlargement of adipocytes, characterized by an increase in large-sized adipocytes and a decrease in small-to-medium-sized adipocytes. Treatment with HHT partially mitigated the hypertrophic expansion of the adipocytes, indicating improved WAT function (Fig. 3G). In addition, the levels of proinflammatory cytokines (IL-1β, MCP1) and their regulator (AP1)35,36 were increased by HF, while HHT treatment suppressed this proinflammatory response (Fig. 3H). At the protein level, key components of the SASP involved in adipose tissue inflammation and fibrosis which were significantly increased by HF (TGF-β1, MMP2, and PAI-1) were reduced by HHT treatment (Fig. 3I). Protein levels of HSL and ATGL, lipolytic enzymes involved in triglyceride (TG) hydrolysis and insulin sensitivity which are known to be reduced in aged subjects37, were also decreased by HF but normalized by HHT treatment (Fig. 3J). These results suggest that HHT attenuates HF-induced WAT inflammation and metabolic dysfunction.

To test whether HHT impacts thermogenesis, we examined the energy expenditure of the HHT-treated mice (Figs. S7A–C). We found that HHT treatment marginally (p=0.056) increases the energy expenditure (Fig. S7C) and reduces the size of lipid droplets in brown adipose tissue (BAT) with significantly increased uncoupling protein (UCP1) expression in BAT (Figs. S7D and S7E). These data suggest that a moderate increase in energy expenditure by activation of BAT partially contributes to metabolic improvement by HHT treatment.

Taken together, these results suggest that HHT reduces adipose tissue inflammation and improves the thermogenic and lipolytic functions of adipose tissue, likely through the elimination of senescent cells in HF-fed obese mice.

Single nucleus RNA sequencing analysis reveals that HHT attenuates adipocyte senescence in WAT of HF-fed obese mice
WAT is a highly heterogeneous endocrine organ comprising various cell populations, including mature adipocytes, APCs, immune cells, and vascular cells38,39. Therefore, to determine the main target cell populations that contribute to the healthy remodeling of WAT and improved metabolic homeostasis by HHT treatment, we performed single nucleus RNA sequencing (snRNA-seq) analysis40,41. The epididymal WAT of three mice from each group were pooled and processed. Unsupervised clustering based on differentially expressed genes (DGEs) revealed distinct WAT cell populations that are projected onto 14 different clusters in both PBS- and HHT-treated adipose tissues, as illustrated in the t-distributed stochastic neighbor embedding (tSNE) plots (Fig. 4A). Similar to previous reports40,41, these include mature adipocytes, APCs, macrophages, T- and B-cells, and vascular endothelial cells. The heatmap for the top three genes of each cluster demonstrates the cell-type-specific transcriptome signature (Fig. S8A). The stacked barplot for the proportion of each cluster demonstrates the impact of HHT treatment on the frequency of each cell population (Fig. 4B). We confirmed the expression of cell-type-specific genes for major cell populations, including adipocytes (Cluster 5), APCs (Cluster 2), mesothelial cells (Cluster 8), macrophages (Cluster 0, 1, 3, 7), T-cells (Cluster 4), and B-cells (Cluster 6) (Fig. S8B).

Notably, the proportion of adipocytes (Cluster 5) increased in the HF-HHT-treated WAT compared with the HF-PBS-treated WAT (Fig. 4A, B). This result, along with reduced adipocyte sizes (Fig. 3E, G), suggests increased hyperplastic adipogenesis (i.e., adipocyte cell number increase), which is closely associated with improved WAT metabolic function and enhanced glucose homeostasis. To gain insights into the HHT-mediated molecular changes in adipocytes, we performed DGE analysis between the HF-HHT- and HF-PBS-treated adipocytes and found significantly altered genes presented in the volcano plot (Fig. 4C middle panel). Subsequent gene ontology (GO) enrichment analysis of DGEs in the HHT-treated adipocytes exhibited enriched signatures associated with insulin sensitivity (e.g., Adipor2), lipid and carbohydrate catabolic process, and fat cell differentiation (Fig. 4C, right panel; HHT>PBS plot). In contrast, the PBS-treated adipocytes showed signatures of cell migration and tissue organization (Fig. 4C, left panel; PBS>HHT plot). We further characterized the functional aspect of mature adipocytes (Cluster 5) and found a positive correlation of HHT-treated adipocytes with healthy adipokine profiles, such as Adipoq (Adiponectin), Cfd (Adipsin), and Vegfa (VEGF-A) (Fig. 4D)42–45. Next, to directly test whether HHT treatment reduces adipose tissue senescence, we analyzed the SenMayo gene set, a panel of 125 genes that are associated with cellular senescence46. We examined the SenMayo signature scores in various cell types and observed that the score was significantly lower in adipocytes and myeloid/erythroid cells, while it was increased in macrophages with higher M1-like and lower M2-like signature (Fig. 4E, S8C and S8D). This finding is unexpected and somewhat inconsistent with our histopathological data, which showed a reduced crown-like structure (CLS) following HHT treatment (Fig. 3E). Despite the seemingly contradictory transcriptomic signatures, our immunofluorescent staining with the M1-like macrophage marker iNOS further confirmed that CLS with iNOS-positive pro-inflammatory macrophages was significantly reduced in HHT-treated WAT (Fig. S8E). These findings indicate that HHT attenuates adipocyte senescence and may exert differential effects across various cell types. Furthermore, HHT-treated adipocytes exhibited significantly lower γ-H2AX immunofluorescent signal, indicating lower levels of DNA damage (Fig. 4F). Taken together, these data illustrate that HHT suppresses adipocyte senescence and improves the metabolic function of adipocytes.

HHT improves APC plasticity and metabolic function in WAT of HF-fed obese mice
Our snRNA-seq demonstrated an increase in adipocyte number in HHT-treated adipose tissue (Fig. 4A, B). Increased adipocyte number is directly linked with the improved metabolic function of WAT and is mediated through the expansion and differentiation of APCs, which are significantly reduced in age-associated WAT47,48. To explore the effect of HHT on APCs, we performed DGE analysis between the HF-HHT- and HF-PBS-treated APCs and found an increased signature of ECM organization and tissue adhesion in the APC population in the PBS-treated APCs (Cluster 2) (Fig. 5A, left panel: PBS>HHT plot). The HF-PBS-treated APC showed enriched expression of collagen genes (Col5a2 and Col4a1) compared with the HF-HHT-treated APCs (Fig. 5A, middle volcano plot). Our GO analysis revealed that the collagen biosynthesis process was significantly reduced by HHT treatment in several cell clusters, including adipocytes (Cluster 5) and APCs (Cluster 2), two major players of adipose fibrosis (Fig. 5B)15,49. Notably, we found that HHT significantly reduces the intensity of fibrosis-specific staining (Fig. 5C). Since adipose fibrosis is closely related to a decline of the adipogenic differentiation of APCs14,15, we tested the effect of HHT on the differentiation of APCs using a senescent 3T3-L1 differentiation model (Fig. S9A). Senescent 3T3-L1 preadipocytes lost their adipogenic differentiation capacity, and HHT treatment restored this capability, as confirmed by oil red O (ORO) staining (Fig. S9B). Moreover, HHT treatment reduced senescence phenotypes, such as reduced SAβG-positive cells and p16 expression, of 3T3-L1 cells (Figs. S9C and S9D). Together, our results demonstrate that HHT treatment enhances the adipogenic differentiation of APCs. APCs’ cellular plasticity, such as cold-induced beige adipocyte formation, has been shown to decline with age14,50. To investigate the effect of HHT on cold-induced beige adipogenesis, we kept the mice in a cold (4 °C) environment (Fig. 5D). The expression of cold-induced beige-specific genes was significantly increased in HHT-treated mice compared with the PBS-treated group (Fig. 5E). This indicates that HHT restores the potential of APCs to form cold-induced beige adipocytes, which is otherwise lost in metabolically unhealthy adipose tissue14.

Finally, to test whether the improved whole-body glucose handling observed with HHT treatment is a result of improved metabolic function of the adipose tissue, we performed hyperinsulinemic-euglycemic clamp studies. HHT administration restored the whole-body glucose turnover that was decreased by HF (Fig. 5F). We also found increased glucose uptake in the HHT-treated WAT compared with the PBS-treated WAT (Fig. 5G), indicating enhanced insulin sensitivity in WAT following HHT treatment. Additionally, we examined liver tissues and found significantly reduced steatosis in the HHT-treated liver with decreased TG accumulation and alanine aminotransferase (ALT) enzyme (Fig. 5H–J). These findings indicate that HHT-mediated enhancement of APC plasticity, along with reduced adipocyte senescence, promotes healthier adipose tissue remodeling and metabolic homeostasis in HF-fed obese mice.

HHT improves adipose tissue senescence and insulin resistance in aged mice
Next, to examine the effect of HHT on age-associated insulin resistance and adipose tissue dysfunction, aged mice (18 months) fed with a normal chow diet were administered PBS or HHT three times a week. Body weight and accumulated food intake were not significantly different between the groups (Fig. 6A, B). However, the cumulative weight gain was significantly lower in the HHT-treated mice, leading to a reduced food efficiency ratio in the HHT group (Fig. 6C, D). Importantly, HHT treatment improved blood glucose clearance and insulin sensitivity (Fig. 6E–H) in the aged mice. Furthermore, the percentages of SAβG-positive cells and adipocyte size were reduced in the HHT-treated WAT (Fig. 6I and S10A). Additionally, the levels of p21 and p16 proteins were significantly reduced in HHT-treated WAT compared to PBS-treated WAT (Fig. 6J). The levels of precursor IL-1β, p-p65/p65 ratio, TGF-β1, MMP2, and PAI-1 proteins were also significantly reduced by HHT treatment (Fig. 6K and S10B). The biological age of WAT in HHT-treated mice was also assessed using epigenetic age and was observed to be lower in HHT-treated mice (Fig. 6L). These results suggest that HHT attenuates adipose tissue senescence and improves metabolic disturbance in aged mice.

We showed the senotherapeutic effect of HHT in human preadipocytes, suggesting its potential impact on human WAT. Thus, we further tested the effect of HHT on cellular senescence ex vivo in human WAT. As expected, HHT treatment decreased SAβG staining in human WAT in a dose-dependent manner (Fig. 6M). Consistent with this, HHT decreased the SAβG-positive cells in the tissue sections of the human WAT samples (Fig. S10C) and the levels of p53 and p21 proteins (Fig. 6N) in a dose-dependent manner. These findings indicate that HHT has senotherapeutic activity in human adipose tissue.

HHT exerts senotherapeutic effects by targeting HSPA5
To explore a potential target of HHT, we employed a Drug Affinity Responsive Target Stability (DARTS) approach51 followed by mass spectrometry. DARTS is based on the principle that the binding of a small molecule to a target protein stabilizes the protein’s structure and makes it protease resistant (Fig. 7A). We initially performed DARTS using RS HDFs and observed many proteins that are resistant to protease (i.e., pronase) upon HHT treatment (Fig. S11A). Among them, we selected 25 pronase-resistant protein bands on several gels, which might bind putatively with HHT. With mass spectrometry, we then identified 12 candidate proteins (Supplementary Table 4). We further confirmed by Western blot whether these 12 candidate proteins are resistant to pronase through binding with HHT, and identified the stability of 5 proteins: PKM2, HSPA5, HSPA8, TXNRD1, and RPL7, upon HHT treatment (Fig. 7B and S11B). To identify the target proteins that mediate the senotherapeutic activity of HHT, each candidate protein was knocked down in RS HPAs with siRNAs (Fig. S11C), and then the senotherapeutic activity of HHT was tested. Among them, the knockdown of HSPA5 (heat shock protein family A member 5) abolished HHT-induced cytotoxicity, which was accompanied by reduced Casp-3 levels (Fig. 7C–E). Conversely, we overexpressed HSPA5 in HPAs and found that the senotherapeutic activity of HHT was enhanced in HSPA5 overexpressed cells (Fig. 7F and S11D). HSPA5 protein levels were consistently higher in senescent cells than in NS cells across HPAs, HDFs, and hRPEs (Fig. S11E). To further confirm whether the senotherapeutic activity of HHT might be mediated through HSPA5, NS and RS HPAs were treated with 100 nM HHT for 2 days–a time window selected to capture the induction and progression of apoptosis without causing overt cell death–and subsequently subjected to co-immunostaining for HSPA5 and Casp-3, using DAB (brown) and Fast Red (red) as chromogens, respectively. RS HPAs treated with HHT consistently showed higher expression of HSPA5 and Casp-3 than HHT-treated NS HPAs (Fig. 7G). Among HHT-treated RS HPAs, cells with high HSPA5 expressions showed a significantly greater number of Casp-3-positive cells compared with those expressing low HSPA5 (Fig. 7H). These data suggest that HSPA5 high expressing cells are primary target of HHT, and HSPA5 may serve as a key mediator of the senotherapeutic activity of HHT. We also prepared HPAs in three distinct states of cellular senescence through serial passaging: low passage (p2-p3), mid passage (p6-p7), and high passage (p12-p13). As passage number increased, the expression of HSPA5, p53, p21, and p16 proteins increased and the senotherapeutic effect of HHT was enhanced with the elevated expression of HSPA5 (Figs. 7I, J, and S11F–H).

To directly assess the physical interaction between HHT and HSPA5, we conducted a Surface Plasmon Resonance (SPR) assay, which allows for the measurement of HHT’s affinity for HSPA5 protein52. The SPR assay revealed a significant molecular affinity between HHT and HSPA5 (Fig. 7K). To further investigate the molecular binding between HHT and HSPA5, we employed a binding model prediction. The initial binding model was determined using rigid docking, followed by Molecular Mechanics energies combined with the Generalized Born and Surface Area (MM-GBSA) calculation, a widely used method for estimating the free energy of the binding of small molecules to protein53. HSPA5 is known to play a pivotal role in cell survival, and suppression of HSPA5 by inhibitors that bind to the ATP binding site of HSPA5 can lead to cell death54. Therefore, we postulated that HHT may inhibit the function of HSPA5 through binding to the ATP binding site. The predicted binding model of HSPA5 and HHT shows key interactions including four hydrogen bonds (H-bonds) and one pi-cation interaction with HSPA5 residues (Fig. 7L and S11I). These results suggest that HHT can bind to HSPA5 and may act as an inhibitor of HSPA5. To test this hypothesis, we measured the ATPase activity of HSPA5 in the presence of HHT or HA15, a potent and specific inhibitor of HSPA555. We found that the inhibitory activity of HHT was greater than HA15, demonstrating that HHT acts as an HSPA5 inhibitor by reducing its ATPase activity (Fig. 7M). Our data collectively suggest that HHT physically interacts with HSPA5 to inhibit its ATPase activity and induce cytotoxicity in HSPA5 high-expressing senescent cells, subsequently leaving HSPA5 low-expressing cells.

In metabolic tissues, HSPA5 protein levels were significantly increased in both adipose tissue and skeletal muscle from HF-induced obese mice compared with chow-fed controls, whereas its expression was not significantly changed in the liver (Fig. 7N and S11J). The increase in HSPA5 expression was more prominent in adipose tissue than in skeletal muscle, suggesting that adipose tissue is a primary metabolic target organ responsive to HHT treatment. Indeed, HHT treatment in HF-fed obese or aged mice resulted in reduced levels of HSPA5 protein in adipose tissues due to the elimination of HSPA5 high-expressing senescent cells (Fig. 7N–P), providing mechanistic insight into the senescent cell targeted activity of HHT. In addition to its action through HSPA5, HHT is also known to inhibit the translation of ribosomal protein56, which may be involved in mediating the senotherapeutic effect of HHT. Thus, to test this possibility, we examined the expression of ribosomal protein, RPS14, which is known to induce cell cycle arrest and senescence57. No significant alteration in the level of RPS14 protein was observed in either non-senescent or senescent cells treated with HHT (Fig. S12A). In addition, we tested whether other translation inhibitors, such as cycloheximide (CHX), anisomycin (Aniso), and puromycin (Puro), influence the senescence of RS HPAs. We observed that none of the three translation inhibitors altered cell numbers, expression of aging biomarkers, and SAβG staining in RS HPAs (Figs. S12B–D). These findings indicate that the senotherapeutic activity of HHT is not associated with its translation-inhibitory function in HPAs. Taken together, these data suggest that HSPA5 may be the target molecule mediating the senotherapeutic activity of HHT, independent of HHT’s ribosomal protein inhibition activity.

HHT improves aging phenotypes and extends lifespan in progeroid mice
To further evaluate the effect of HHT on aging phenotypes and lifespan, we employed a Zmpste24-/- progeroid mice, an animal model for Hutchinson-Gilford progeria syndrome58. HHT administration improved physical activity in Zmpste24-/- mice (Supplementary Movie 1). HHT treatment significantly extended lifespan, increasing the median lifespan from 14 to 15 weeks and the maximum lifespan from 15 to 19 weeks (Fig. 8A). HHT also increased bone mineral content, bone area, lean body mass (Figs. 8B, C, and S13A), and grip strength (Fig. 8D) in Zmpste24-/- mice. To determine whether HHT administration improves pulmonary function, a key aspect of the senescent phenotype in this model59, we conducted non-invasive whole-body plethysmography and found that airway resistance measured by enhanced pause (Penh) was significantly decreased in Zmpste24-/- mice (Fig. 8E). Histological analysis revealed that HHT treatment decreases collagen deposition (Fig. 8F), the percentages of SAβG staining, and p53 positive cells (Fig. 8G) in the lung tissues of Zmpste24-/- mice. In addition, HHT reduced the levels of senescence-related proteins (p53, p21, and p16), which are increased in the lung tissues of Zmpste24-/- mice (Fig. 8H). These results suggest that HHT treatment mitigates aging phenotypes in progeroid mice.

Mice often develop lymphoma, which is among the leading causes of death in aged mice60,61. As HHT has been used for the treatment of hematological oncology, the extended lifespan may primarily be due to the anti-cancer effects of HHT rather than its senotherapeutic effects. However, we did not find any differences in blood smear (Fig. S13B), CBC profiles (Supplementary Table 5), the numbers of Peyer’s patches (Fig. S13C), and the weight of inguinal lymph nodes (Fig. S13D) among Zmpste24+/+, Zmpste24+/-, and Zmpste24-/- mice treated with HHT or vehicle (PBS) control. Additionally, no significant histological alterations were observed in the Peyer’s patches, inguinal lymph nodes, or the spleen (Fig. S13E). These results suggest that HHT mitigates aging phenotypes and extends lifespan in the absence of tissue toxicity in progeroid mice, independent of lymphocyte suppression.

HHT delays aging and extends lifespan in aged mice
Given these beneficial effects of HHT on metabolic function in the aged mice, we postulate that HHT also impacts general health and longevity of aged mice. To test this, we treated 16-month-old mice with HHT once per week over a period of 12 months (Fig. 9A). We found that HHT significantly extended the lifespan and increased physical activity of aged mice (Fig. 9B and Supplementary Movie 2). This was accompanied by improved kidney function, represented by reduced proteinuria (Fig. 9C, D). It also improved skeletal muscle function measured by grip strength (Fig. 9E). In addition, HHT reduced fibrosis of the kidney, lung and liver in aged mice (Fig. S14A). HHT is primarily metabolized by the liver and excreted in the urine through the kidneys62,63. Thus, to test the potential adverse effects of prolonged treatment with HHT on these organs, we measured the levels of liver enzymes (i.e., aspartate aminotransferase (AST) and ALT), creatinine, and urea nitrogen (UN). We did not find any differences between PBS- and HHT-treated groups (Figs. S14B-G). Furthermore, as suppression of hematopoiesis is observed in HHT-treated cancer patients63,64, we examined the number of circulating hematopoietic cells and spleen histology, and again found no difference between control and long-term HHT-treated mice (Fig. S14H and Supplementary Table 6). Collectively, our data demonstrate that HHT delays aging and extends lifespan in mice without major adverse effects.

As obesity-induced metabolic impairment expedites the aging process and exacerbates age-related diseases65,66, we employed an additional aging model by a combination of HF and aging. Two-month-old mice were fed a HF diet for 11 months, and HHT was administered by once-weekly injections for 5 months while continuing the HF regimen (Fig. 9F). We found that HHT significantly extended the lifespan of mice, increasing the median lifespan from 509 to 551 days, whereas the maximum lifespan could not be determined because the mice were sacrificed at 558 days for tissue collection (Fig. 9G). In addition, HHT induced weight loss, mainly attributable to fat rather than lean mass reduction (Fig. 9H, I). To investigate the senotherapeutic effects of HHT on various cellular populations in WAT, we performed mass cytometry (CyTOF) analysis39,67 (Fig. S15A). Intriguingly, despite no significant differences in the number of APCs in perigonadal WAT (pWAT), we found reduced expression of CD142 and ICAM-1, markers for committed APCs, in HHT-treated WAT, indicating that HHT-treated APCs maintain characteristics of stemness39,67 (Fig. 9J, K). However, we did not find any differences in the adipose tissue immune cell populations, except for the reduction in M1-like macrophages (Fig. S15B), which is consistent with our data shown in Figs. 3E and S8E. Moreover, we did not find any differences in the histopathology of spleen tissue, blood smear, or the number of splenic lymphocytes between PBS- and HHT-treated mice, suggesting that the extended lifespan is not associated with the lymphoma suppression activity of HHT, and that HHT treatment does not cause toxicity to hematopoietic cells, liver, small intestine, or kidney (Figs. S15C–G). We found moderately reduced proteinuria in HHT-treated mice without signs of renal toxicity (Figs. S15H–M). Collectively, these results indicate that HHT delays aging and prolongs lifespan in physiological aging mice.

Methods
Ethical approval for animal studies and human adipose tissue research
Animal studies and human adipose tissue research were conducted in compliance with all relevant ethical regulations. Animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Yeungnam University College of Medicine (YUMC-AEC2019-031, YUMC-AEC2020-025, and YUMC-AEC2022-039) and by the Animal Care Committee of The Centre for Phenogenomics (TCP, AUP#27-0271H). Research involving human adipose tissues was approved by the Institutional Review Board (IRB) of Yeungnam University Medical Center (YUMC 2021-03-052).

Materials
Clinically applied compounds (2150 compounds) under phase 1 to 3 clinical trials and FDA-approved were kindly provided by Korea Chemical Bank (Daejeon, Republic of Korea) (https://chembank.org/). Cell Counting Kit-8 (CCK-8), and lactate dehydrogenase (LDH) assay kit were obtained from Dojindo Molecular Technologies (Rockville, MD, USA), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) from Amresco LLC (Solon, Ohio, USA), doxorubicin from Ildong Pharmaceutical Co. Ltd. (Seoul, Republic of Korea), and HHT from Tocris (Bristol, UK). Dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), and insulin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies used in this study for Western blotting, immunostaining, and mass cytometry are listed in Supplementary Table 7 and 8. Sequences for primers used in RT-qPCR are listed in Supplementary Table 9. siRNAs were purchased from Life Technologies Corp. (Carlsbad, CA, USA), and detailed information is provided in Supplementary Table 10. HPAs (human visceral preadipocytes, #PT-5005) and HUVECs (human umbilical vein endothelial cells, #C2517A) were purchased from Lonza (Basel, Switzerland). HDFs (human dermal fibroblasts, #PCS-201-012), hRPEs (human retinal pigment epithelial cells, #CRL-2302), and 3T3-L1 mouse preadipocytes (#CL-173) were purchased from American Type Culture Collection (Manassas, VA, USA).

Cell culture
HDFs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Welgene Inc., Gyeongsan, Republic of Korea) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), while HPAs were maintained in a preadipocyte basal medium with appropriate supplements and growth factors (Lonza, Basel, Switzerland) containing 10% FBS. HUVECs were grown in endothelial cell basal medium-2 enriched with various growth factors, and hRPEs were cultured in DMEM:F-12 medium with 10% FBS. Additionally, 3T3-L1 cells were maintained in DMEM with 10% calf serum (Gibco).

Preparation of prematurely senescent cells by doxorubicin treatment
To induce premature senescence (PS), HDFs and hRPEs were treated with 0.5 μM doxorubicin in serum-free media for 4 h99. Then, cells were cultured with DMEM (10% FBS) and DMEM/F-12 (10% FBS) for 4 days and induction of cellular senescence was confirmed by measuring the upregulation of p16 and SASP levels as well as an increase in SAβG staining. The percentages of SAβG staining in PS HDFs and PS hRPE were more than 70% and 60%, respectively.

Preparation of replicatively senescent cells
Replicatively senescent (RS) model was established following the protocol outlined in previous studies99,100. Briefly, HDFs (2 × 105), HUVECs (2 × 105), and HPAs (2 × 105) were cultured in a 100 mm dish and sub-cultured when cells reached 80-90% confluency. The number of population doublings (PDs) was calculated from the geometric equation: PD=log2 F/log2 I, where F is the cell number at the end of incubation time, and I is the cell number at the beginning of the incubation time. We also measured the population doubling time (PDT), which was calculated using the following equation: PDT=(Tf-Ti)/log2 (F/I), where Tf is the final incubation time, Ti is the initial incubation time, F is a final population number, and I is an initial population number. For all experiments, HDFs were used after passage 5 (PD < 20, PDT<2 days) or passage 23 (PD > 60, PDT>14 days). HUVECs were used in passage 5 (PD < 20, PDT<2 days) or passage 23 (PD > 60, PDT>14 days). HPAs were used in passage 3 (PD < 20, PDT<2 days) or passage 13 (PD > 60, PDT>14 days). These are referred to as “non-senescent (NS)” and “senescent” cells, respectively. Cellular senescence in RS HDFs, RS HPAs, and RS HUVECs was confirmed by measuring the upregulation of p53, p16, and SASP levels and an increase in SAβG staining. The percentages of SAβG staining in RS HDFs and RS HUVECs were more than 80% and RS HPAs were more than 60%.

Cell viability assay
Cell number was assessed using the CCK-8 according to the manufacturer’s instructions and by manual cell counting under light microscopy.

Lactate dehydrogenase (LDH) assay
LDH activity was measured in media using an LDH assay kit according to the manufacturer’s instructions.

Senescence-associated β-galactosidase (SAβG) staining assay
SAβG activity was measured with minor modifications100. Cells were washed 2 times with phosphate-buffered saline (PBS) and fixed with 3.7% (v/v) formaldehyde in PBS for 5 min. The human adipose tissues and the epididymal WAT of mice were fixed in 4% paraformaldehyde (PFA) for 30 min and washed twice with PBS. Cells and tissues were incubated in a SAβG staining solution (1 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-galactoside, 40 mM citric acid-sodium phosphate (pH 6.0), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 150 mM NaCl, and 2 mM MgCl2) for 18 h at 37 °C. Tissue samples were embedded with an O.C.T compound and cryosectioned to 10 μm at -30 °C. SAβG positive cells stained with blue color were counted and analyzed.

Screening of senotherapeutic candidates from FDA-approved drugs
To identify senotherapeutic candidates from a library of 2150 FDA-approved drugs, primary and secondary screenings were conducted. In the primary screening, PS HDFs were seeded at 1 × 10³ cells per well in a 96-well plate and cultured overnight. The cells were then treated with each drug at a concentration of 100 nM and incubated for 4 days at 37 °C in a CO2 incubator. Cell viability was assessed using the CCK-8 assay, while cellular senescence was evaluated by SAβG staining. Based on these results, 110 compounds were selected for further analysis. In the secondary screening, RS HDFs, RS HUVECs, and PS hRPEs were seeded at 1 × 10³ cells per well in a 96-well plate, cultured overnight, and treated with each drug at 100 nM for four days. Cell viability was assessed using the CCK-8 assay, and cellular senescence was determined by SAβG staining. To evaluate potential toxicity, non-senescent cells were also treated with 100 nM of each compound for four days, followed by a viability assessment using the CCK-8 assay. ABT263 and rapamycin were used as positive controls for senolytic and senomorphic activity, respectively.

Cell cycle analysis by flow cytometry
Cells were harvested and fixed in 70% cold ethanol at 4 °C for 1 h. Cells were washed twice with PBS and resuspended in a propidium iodide (50 µg/mL, Sigma-Aldrich, P4170) solution. RNase A was added to a final concentration of 0.5 µg/mL, and the samples were incubated at 4 °C for 4 h. Flow cytometric analysis was performed using a CytoFLEX Flow Cytometer (Beckman Coulter Life Sciences, Brea, CA, USA).

Adipocyte differentiation of 3T3-L1 cells
3T3-L1 preadipocytes were cultured in DMEM with 10% calf serum until two days post confluence (Day 0). To induce differentiation, the cells were treated with MDI differentiation medium, consisting of DMEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 0.5 μg/mL insulin. On Day 2, the medium was replaced with a maturation medium composed of DMEM containing 10% FBS and 0.5 μg/mL insulin. The cells were maintained in this medium for an additional 6 days, with medium changes every 2 days. On Day 8, adipocyte differentiation and cellular senescence were assessed using Oil Red O staining, SAβG staining, and Western blot analysis.

Oil red O (ORO) staining
Cells were fixed with 4% paraformaldehyde for 30 min, rinsed 2 times with PBS, and incubated with an ORO working solution for 1 h. The images were photographed under a microscope, and ORO staining density in cells was quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Transfection of siRNA and plasmids in HPAs
Senescent HPAs were seeded in 60 mm dishes and incubated overnight. siRNAs (20 nM) were transfected into cells using RNAiMax 2000 transfection reagent (Invitrogen, Gaithersburg, MD, USA). Protein knockdowns were confirmed by Western blot analysis. For overexpression study, HPAs were transfected with a pCMV-HSPA5-His plasmid purchased from SinoBiological (Seoul, Republic of Korea) using Lipofectamine 2000 reagent (Invitrogen) and the expression levels of HSPA5 protein were confirmed by Western blotting.

Animals and animal experiments
C57BL/6N mice were purchased from Koatech (Pyungtaek, Republic of Korea), and p16-Luc mice were kindly donated by Eiji Hara (Osaka University, Japan). Zmpste24-/- mice on a C57BL/6J background were kindly gifted by Dr. Zhongjun Zhou (University of Hong Kong, China). To examine the effect of senotherapeutic candidates and HHT on adipose tissue senescence and insulin resistance in obesity, 8-week-old male C57BL/6N mice and p16-Luc were randomly divided into chow diet (Envigo, Indianapolis, IN, USA) or HF groups (Research diets, New Brunswick, NJ, USA) for 6 weeks. HF contains 60.3% of the total calories as fat, 21.3% as carbohydrates, and 18.4% as protein. Then, mice in an HF group were divided into two groups, PBS- or senotherapeutic candidate-treated group, and placed on the same HF for further 6-8 weeks. Senotherapeutic candidates including HHT were dissolved in DMSO and diluted in PBS. Senotherapeutic candidates were injected intraperitoneally three times a week at the indicated dosages (Supplementary Table 2). Mice were given ad libitum access to water and food. To examine the effect of HHT on adipose tissue browning in obese mice, HF-fed mice were exposed to 4 °C for 6 h following PBS and HHT administration for 8 weeks. The HF and drinking water were provided during cold exposure.

To examine the effects of HHT on adipose tissue senescence and insulin resistance in aged mice, 18-month-old male C57BL/6N mice (DBL, Eumseong, Republic of Korea) were randomly divided into PBS- or HHT-treated groups and were administered intraperitoneally with PBS or HHT three times a week for 14 weeks. They were provided with a chow diet.

To measure the lifespan of aged mice, C57BL/6J (16 months old) male mice were intraperitoneally injected once a week with HHT for 12 months or PBS (n=8 in each group). For the lifespan of Zmpste24-/- mice, Zmpste24-/- mice (5 weeks old) were administered intraperitoneally once a week for 14 weeks with HHT or PBS (n=7 for PBS and n=9 for HHT in male, and n=4 for PBS and n=8 for HHT in female mice). For the lifespan of HF-fed aged mice, C57BL/6J (2 months old) male mice were fed an HF for 11 months, and HHT was administered once a week intraperitoneally for 5 months while continuing the HF regimen.

Mice were housed in a controlled environment with a 12h light:12h dark cycle. Food intake and body weight were measured manually once a week during the experimental periods. Mice were anesthetized with 2,2,2-tribromoethanol and tert-amyl alcohol (375 mg/kg), and blood was collected from the orbital plexus. The plasma and tissues were stored at -80 °C until analysis. The in vivo experiments were performed in strict accordance with guidelines and protocols approved by the Institutional Animal Care and Use Committee of Yeungnam University College of Medicine (permit number: YUMC-AEC2019-031, YUMC-AEC2020-025, and YUMC-AEC2022-039) and by the Animal Care Committee of The Centre for Phenogenomics (TCP, AUP#27-0271H).

Ex vivo explant culture of human subcutaneous adipose tissues
This study has been approved by the IRB at the Yeungnam University Medical Center (YUMC 2021-03-052). All patients declared their informed consent in written form. Human subcutaneous adipose tissues were obtained from female patients who underwent mammoplasty after mastectomy due to breast cancer. Detailed information was provided in Supplementary Table 11. Adipose tissue samples were cut into 5×5×5 mm pieces, placed in 48 well plates, and cultivated in DMEM containing 10% FBS and antibiotics at 37 °C in a 5% CO2 humidified incubator. Human subcutaneous adipose tissue samples were treated with the indicated concentrations of HHT twice every 3 days for 7 days.

Whole-body plethysmography
Airway responsiveness was determined through whole-body plethysmography (Buxco Research System, Wilmington, NC, USA). Briefly, mice were placed in a whole-body plethysmography chamber. After a few minutes for stabilization, enhanced pause (Penh, an indication of airway resistance) was determined.

Triglyceride (TG) measurement
The liver was homogenized in a 20-fold solution of chloroform: methanol (2:1) containing 0.01% butylated hydroxyl toluene, and vortexed for about 30 sec. After incubation overnight at 4 °C, the homogenized sample was diluted twice with distilled water and centrifuged at 800 x g for 30 min. The chloroform layer was separated, and the same amount of chloroform containing 1% triton X100 was added. After the evaporation of chloroform, the remaining part was dissolved with distilled water. Triglyceride Reagent and Free Glycerol Reagent (Sigma-Aldrich) were used to measure the level of TG. The concentration was calculated with the standard solution.

Intraperitoneal glucose and insulin tolerance test
An intraperitoneal glucose tolerance test (IPGTT) was conducted after an overnight fasting for 16 h. Before glucose administration, blood was collected from the tail vein to measure basal blood glucose and plasma insulin levels. Animals were injected intraperitoneally with glucose (1 g/kg body weight). Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after injection with blood from the tail vessel, using an ACCU-CHEK Instant glucometer (Roche, Basel, Switzerland). Plasma at 0, 15, 30, and 60 min was stored at -80 °C for insulin measurement. For the insulin tolerance test (ITT), mice were fasted for 6 h and injected with insulin intraperitoneally at 0.75 IU/kg body weight. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after injection using the same glucometer.

Hyperinsulinemic-euglycemic clamp
The hyperinsulinemic-euglycemic clamp study was conducted in conscious mice 4 days after vein cannulation. The vein cannulation surgery was performed under isoflurane inhalation anesthesia, during which an indwelling catheter was inserted into the left internal jugular vein. The catheter was externalized through a small incision in the skin at the back of the neck. Following the surgery, the mice were individually housed and carefully monitored until they regained their pre-operative body weight, indicating full recovery.

After an overnight fasting, hyperinsulinemic-euglycemic clamp was performed with a priming dose (900 pmol/kg body weight) of human insulin (Lilly, IN, Indianapolis, USA), and constant infusion of insulin at a rate of 24 pmol/kg/min, 20% glucose was infused for maintaining glucose at constant concentrations of 5-6 mM through a Y connector for 2 h using a microdialysis pump (CMA Microdialysis, Kista, Sweden). Blood was withdrawn from the tail vessel, and the plasma glucose levels were measured with a glucose analyzer (Analox, Middlesbrough, UK). The insulin-stimulated whole-body glucose uptake rate was estimated with a continuous injection of [3-3H]glucose (PerkinElmer Life and Analytical Sciences, Shelton, CT, USA) throughout the clamps (0.1 μCi/min). The insulin-stimulated glucose uptake rate in epididymal fat was measured by 2-deoxy-D-[1-14C]glucose (2-[14C]DG) administration as a bolus (10 μCi) after starting the clamp at 75 min. After the clamp was over, mice were anesthetized with avertin, and collected plasma and tissue samples were stored at -80 °C for further analysis. To determine tissue 2-[14C]DG 6-phosphate content, tissue samples were homogenized, and 2-DG-6-phosphate was separated from 2-DG in supernatants using an ion-exchange column (Bio-rad, Hercules, CA, USA). Whole body glucose turnover rate was calculated as the ratio of the glucose infusion rate to the specific activity of plasma glucose. The glucose uptake rate in each tissue was calculated from the plasma 2-[14C]DG profile using MLAB (Civilized Software, Silver Spring, MD, USA) and the tissue 2-[14C] DG-6-phosphate content.

Blood and urine assay
Plasma insulin levels were measured with an enzyme-linked immunosorbent assay kit (Merck, Darmstadt, Germany). The liver enzymes, ALT and AST were measured in plasma using the commercial kit (Asan pharm, Seoul, Republic of Korea).

To collect urine, mice were held over the cling wrap and lightly stroked the belly of the animal. Urine was collected into the tube and urine protein assay was performed using a strip test (Medi-Test Combi 10L, Allentown, PA, USA), and BCA assays (Thermo, Meridian, Rockford, IL, USA).

Urea nitrogen and creatinine in urine and serum were analyzed by using Urea Nitrogen Colorimetric Detection Kit (Arbor Assays, Ann Arbor, Michigan, USA) and QuantiChrom™ Creatinine Assay Kit (BioAssay Systems, Hayward, CA, USA), respectively according to the manufacturer’s instruction. Urine total protein, albumin, creatine, potassium, and sodium were analyzed by the Beckman Coulter AU480 Clinical Chemistry Analyzer (Brea, CA, USA).

Complete blood count and blood smear
Complete blood count analysis was performed using an automated hematology analyzer (Sysmex XN-1000 hematology analyzer, Sysmex, Kobe, Japan) in whole blood samples. Blood samples (200 µL) were collected from the orbital venous plexus of mice under isoflurane anesthesia and placed into heparinized sample tubes, and a complete blood count analysis was performed. For blood smear, following the collection of blood from mice in EDTA-containing tubes, blood smear samples were prepared on glass slides and stained using the Wright-Giemsa method101.

Bone density measurement with dual-energy X-ray absorptiometry (DEXA)
Body composition was assessed in Zmpste24-/- mice using a DEXA scanner (iNSiGHT VET DXA, OsteoSys Co., Ltd., Seoul, Republic of Korea). Each mouse was anesthetized with an intraperitoneal injection of 240 mg/kg avertin and placed on the scanner bed in the prone position, with the limbs and tail stretched away from the body. Quantitative data within the region of interest were obtained. Body composition of HF-fed aged mice was analyzed using EchoMRI-100 body composition analyzer (Echo Medical Systems, Texas, USA), which quantifies fat and lean mass in live, non-anesthetized mice.

Grip strength
The forelimb grip strength of mice was measured using a BIO-GS3 grip-strength meter (BIOSEB, Pinellas Park, FL, USA). Mice were placed horizontally onto the grid (dimensions 100×80 mm, angled 20 °), allowed to grip the grid with their forelimbs only, and then gently pulled away backward until they released their grip. Results were averaged over 5 trials.

Histological assessment in tissues
Tissues were immediately fixed in 10% neutral formalin. The fixed tissues were divided into an appropriate size to make a paraffin block. Blocks were cut into 5 μm using Microtome (Leica, Wetzlar, Germany). After attaching 2-3 pieces of cut sections to the glass slide, they were dried and sequentially stained with hematoxylin and eosin step. The sections attached to glass slides were first immersed in xylene for 2 min, followed by sequential rehydration in 100% ethanol for 1 minute, 95% ethanol for 2 min, and 90% ethanol for 2 min. After three brief washes in distilled water, the sections were stained with hematoxylin for 7 min, followed by three additional washes. The slides were dipped in acid-ethanol once and immediately washed. Next, the sections were incubated in sodium sulfate for 5 min and rinsed in running water. Eosin staining was performed by immersing the sections in eosin solution for 3 min, followed by washing for 5 min under running water. These sections were then dehydrated through a graded ethanol series (70% to 100%) with three dips at each step. Finally, the sections were dipped in xylene three times and then mounted with a coverslip. The stained samples were observed under a microscope (Olympus, Tokyo, Japan) at 40× and 100× magnification. Pictures were derived using the DIXIeXcope image S/W program (DIXI Science, Daejeon, Republic of Korea), and the size of adipocytes was estimated by measuring the area of all cells in one field at 100× magnification using the ImageJ program. Crown-like structure (CLS) percentage was analyzed by counting the number of surrounded cells by macrophages at 2 fields and then averaging them in the same samples. Paraffin-embedded tissue sections were stained with Sirius Red (Abcam, ab246832) for Picrosirius Red staining or with a Trichrome Staining Kit (Roche, 06521908001) for Masson’s trichrome staining, followed by light microscopy observation. Blue collagen-positive areas in Masson’s trichome staining were analyzed using an ImageJ software.

Immunofluorescence staining
For γH2AX, F4/80, and p53 staining, paraffin-embedded tissue sections were deparaffinized and subjected to heat-induced epitope retrieval in citrate buffer (pH 6.0) using a pressure cooker. Sections were then incubated with Protein Block (Agilent, X090930-2) or 5% BSA (MP Biomedicals, 160069) in PBS to reduce non-specific binding, followed by overnight incubation at 4 °C with primary antibodies diluted in DAKO Antibody Diluent (Agilent, S302283-2). Rabbit anti-γH2AX (Fortis Life Science, IHC-00059; 1:1500), rabbit anti-iNOS (Abcam, #ab3523; 1:100), and rabbit anti-p53 (Abbkine, ABP0110; 1:200) were used. Corresponding secondary antibodies - donkey anti-rabbit IgG Alexa Fluor 647 (Invitrogen, A32795), goat anti-rat IgG Alexa Fluor 647 (Invitrogen, A48265), and goat anti-rabbit IgG Alexa Fluor 546 (Invitrogen, A11035) - were applied at room temperature. Nuclear counterstaining was performed with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, D9542). To reduce tissue autofluorescence, slides were incubated in Sudan Black B (Sigma-Aldrich, 199664) saturated in 70% ethanol for 25 min at room temperature, then mounted with Vectashield Vibrance anti-fade medium (Vector, H-1700). Whole-slide images were acquired at 20× magnification using an Olympus VS120 scanner equipped with a Hamamatsu ORCA-R2 C10600 digital camera (Evident, Japan).

Immunohistochemistry
Paraffin-embedded tissue sections were deparaffinized, treated with freshly prepared 0.45% H₂O₂ in methanol for 15 min at room temperature in the dark, and subjected to heat-induced epitope retrieval in 10 mM citrate buffer (pH 6.0) at 95–98 °C for 15 min. Cell samples were fixed with 4% paraformaldehyde for 15 min and treated with 3% H₂O₂ for 10 min. Both tissue sections and cell samples were permeabilized with 0.2% Triton X-100 in TBS (tris-buffered saline) for 10 min. Tissue sections were blocked with 5% normal donkey serum containing 1% bovine serum albumin (BSA), and cell samples were blocked with 5% BSA (MP Biomedicals, 160069) in PBS for 1 h. Tissue sections were then incubated with a mouse anti-HSPA5 antibody (Santa Cruz Biotechnology, sc-166490; 1:100) at 4 °C overnight. Cell samples were incubated with a mixture of a mouse anti-HSPA5 antibody (Santa Cruz Biotechnology, sc-166490; 1:100) and a rabbit anti–cleaved caspase-3 antibody (Cell Signaling Technology, #9664S; 1:100) at 4 °C overnight. After three washes with TBS-T (TBS containing 0.05% Tween-20), secondary antibodies were applied for 1 h at room temperature. For tissue sections, a horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Cell Signaling Technology, #7076) was used. For cell samples, a mixture of HRP-conjugated goat anti-mouse antibody (Invitrogen, #31430; 1:50) and alkaline phosphatase (AP)-conjugated goat anti-rabbit antibody (Sigma-Aldrich, A3687; 1:50) was applied. Following final TBS-T washes, color development was performed using enzyme-specific substrate systems: HRP activity was visualized with 3,3′-diaminobenzidine (DAB) substrate (Cell Signaling Technology, #7076, or Dako, K5007) to yield brown staining, and AP activity was visualized with Fast Red TR/Naphthol AS-MX (Sigma-Aldrich, F4648) to yield red staining. Finally, tissue sections and cell samples were counterstained with hematoxylin and mounted. Images were acquired using a KF-PRO-005 slide scanner (KF BIOTechnology for Health, Yuyao, China), and quantification of stained areas or positive cells was performed using ImageJ software (National Institutes of Health).

Metabolic cage analysis
Metabolic assessments of each mouse were conducted for a continuous 48 h period using individual closed metabolic chambers (Oxylet; Panlab, Cornellà, Spain). During the measurement period, all mice were provided ad libitum access to food and water to maintain normal physiological conditions. Oxygen consumption (VO2, mL/min) and carbon dioxide production (VCO2, mL/min) were sequentially measured for 3 min per mouse, rotating through all animals housed in the system. Room air was analyzed for 3 min after completing one cycle of all mice to establish baseline gas composition. All collected data, including VO2, VCO2, and energy expenditure, were analyzed using the manufacturer-provided software (Metabolism; Panlab). To account for inter-individual differences, all values were normalized to body weight.

Epigenetic age analysis
The genomic DNA of adipose tissue was prepared using a NucleoSpin DNA Lipid Tissue Kit (Machery-Nagel, Duren, Germany), and the identification of differentially methylated CpG (DMCs) using reduced representation bisulfite sequencing (RRBS) data with 500 ng of genomic DNA.

Briefly, the genomic DNA was digested with MspI and ApeKI and then purified using the Qiagen MiniElute PCR Purification Kit. The DNA fragments were directly subject to end-blunting followed by adding a single adenine nucleotide (dA-tailing) to prepare the DNA for adapter ligation. Methylated adapters were ligated to the prepared DNA, and the adapter-ligated fragments, specifically those within the size range of 160 to 420 bp, were isolated and used for bisulfite conversion utilizing the ZYMO EZ DNA Methylation-Gold Kit™, adhering to the manufacturer’s guidelines. The conversion process alters unmethylated cytosines to uracils, enabling subsequent methylation analysis. Following bisulfite treatment, the libraries were amplified via PCR using PfuTurbo Cx Hotstart DNA polymerase (Agilent Technologies, Santa Clara, CA, USA), a step that enriches the library for subsequent sequencing. The final RRBS libraries were evaluated for quality and size distribution using an Agilent 2100 Bioanalyzer (Agilent Technologies).

The RRBS libraries were sequenced on an Illumina NextSeq 500 (NextSeq 500, Illumina, San Diego, CA, USA).

Sequencing adapters and low-quality bases present in the raw reads were trimmed using the Skewer tool102. Following the quality control and adapter trimming, the resulting high-quality reads were aligned to a reference genome that had been converted to a 3-letter code (to represent bisulfite-treated DNA) using the bs_seeker2-align module of BSseeker2103. After alignment, the bs_seeker2-call_methylation module of BSseeker2 was employed to determine the methylation status at CpG sites across the genome based on the mapped reads with a minimum coverage of 5 to obtain a detailed profile of methylation patterns, enabling the identification of methylation levels at individual CpG sites across the samples in control and treatment groups.

To estimate epigenetic age (DNAm age), we employed a multi-tissue epigenetic age clock model that utilizes ridge regression model for parameter estimation, leveraging DNA methylation data across various tissues. The parameters for this model, including beta coefficients (weights) and intercepts for methylation CpG sites, were sourced from supplemental information reported in the manuscript104. These parameters are well-validated and have demonstrated robustness in predicting chronological age from methylation data across different tissues, including adipose tissue. For each sample in our dataset, epigenetic age was calculated by summing the products of methylation levels at specific CpG sites and their corresponding beta coefficients. Recognizing the challenges associated with the variable number of clock sites available in RRBS, and to enhance the accuracy and consistency of our age estimates, we imputed missing methylation data by filling zero coverage sites with the mean methylation level observed for those sites within the control or treatment groups105. This method ensures a more robust estimation of DNAm age by maintaining a comprehensive dataset, thereby reducing the bias from missing data. The resulting calculation provides a weighted sum of methylation levels, representing an estimation of the epigenetic age in months.

Protein extraction and Western blotting
Cells were washed with ice-cold PBS and harvested by scraping in 50 μl of ice-cold radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl, pH 7.4, 150 mM KCl, 5 mM NaF, and 1 mM phenylmethylsulfonyl fluoride). Cells were ruptured by vortexing two times for 30 sec at 30 min intervals on ice and centrifuged at 16,000 x g for 15 min at 4 °C. To measure SASP in culture media, the culture media were filtered with an Amicon® Ultra-4 Centrifugal Filter, 50 kDa MWCO (Merck KGaA, Darmstadt, Germany) and concentrated using an Amicon® Ultra-4 Centrifugal Filter, 3 kDa MWCO (Merck KGaA). Human adipose tissues were homogenized in 5 volumes of ice-cold RIPA buffer and centrifuged at 16,000 x g for 15 min at 4 °C. Protein concentrations in the supernatants were quantified by the bicinchoninic acid method (Pierce Biotechnology Inc., Rockford, IL, USA). Mouse adipose and lung tissue samples were homogenized in 10 volumes of ice-cold lysis buffer containing 50 mM HEPES, 150 mM NaCl, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamide, 1 mM EDTA, 1 mM EGTA, 1 mM sodium ortho-vanadate, 0.22% β-glycerophosphate, 1% NP40, 10% glycerol, and protease inhibitor cocktail (Santa Cruz Biotechnology., Santa Cruz, CA, USA). The tissue homogenate was centrifuged at 16,000 x g for 10 min at 4 °C, and the supernatant containing protein was collected. Protein concentrations were determined using the Bradford (Bio-rad) assay. An equal amount of protein samples was separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis at 70 V, and then resolved proteins were transferred to a membrane. Membranes were blocked in 1× TTBS (10 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.05% Tween-20) containing 5% skim milk for 30 min at room temperature. Primary antibodies were applied overnight at 4 °C, and then HRP-conjugated secondary antibodies were applied for 90 min at room temperature. After rinsing the membranes 3 times with 1× TTBS for 30 min, antigen-antibody complex was detected using Luminol Reagent (Santa Cruz Biotech Inc). Proteins were visualized with the Fujifilm LAS-3000 image system (Stamford, CT, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control for protein loading. The relative intensities of protein bands, as compared with that of the respective GAPDH signal, were determined by using the Multi Gauge software, version 3.0 (Fujifilm Corp., Stamford, CT, USA), and normalized to 1.0 for control cells by averaging three separate experiments. All phosphorylated forms were normalized to their respective protein forms.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR)
Total RNA was extracted from 80 mg tissue samples using RNeasy Lipid Tissue Mini Kit (QIAGen, Hilden, Germany) according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of RNA using a reverse transcription kit (Applied Biosystems, Foster City, CA, USA). RT-qPCR was performed using the Real-time PCR 7500 System and Power SYBR Green PCR Master Mix (Applied Biosystems). The expression levels of GAPDH were used for sample normalization. Each reaction mixture was incubated at 95 °C for 10 min followed by 45 cycles of 95 °C for 15 sec, 55 °C for 20 sec, and 72 °C for 35 sec.

Single nucleus RNA sequencing and data analysis
For adipose tissue single nuclei sequencing (snRNA-seq), frozen tissues were chopped into smaller pieces on dry ice. Chopped tissues were immediately incubated in lysis buffer before homogenization in glass douncers. Isolated nuclei were then washed twice with cold wash and resuspension buffer. Nuclei suspension was filtered through 40 μm filter before proceeding to the next step. Nuclei were stained with DAPI and flow-sorted. After sorting, nuclei were washed and resuspended for counting. Nuclei were stained with SYBR Green II and counted under the microscope. Sorted nuclei were used as input into the 10x Genomics single-cell 3’ v3.1 assay (Pleasanton, CA, USA) and processed as described by the protocol provided by the 10x Genomics. For sequencing, the molarity of each library was calculated based on library size as measured by bioanalyzer (Agilent Technologies) and qPCR amplification data (Roche). Samples were pooled and normalized to 1.5 nM. The library pool was denatured using 0.2 N NaOH for 8 min at room temperature and neutralized with 400 mM Tris-HCl. Library pool at a final concentration of 300 pM was loaded to sequence on Novaseq 6000 (Illumina). Samples were sequenced with the following run parameters: Read 1–28 cycles, Read 2–90 cycles, index 1–10 cycles, index 2–10 cycles. Sequencing target reads for each library were 40,000–50,000 reads/nuclei.

The 10x Genomics’ CellRanger v5.0.0 software was used for sequence alignment and barcode processing with the mouse reference genome (mm10-2020-A). The filtered gene-count matrices from CellRanger were used for subsequent preprocessing. Doublets were identified and removed through the automated ‘scDblFinder’ toolkit using default parameters106. Low-quality nuclei were removed from the dataset by retaining only nuclei with greater than 400 unique features (genes), less than the 95th percentile of total UMI counts & features, and less than 0.5 percent of mitochondrial transcripts detected. The percentage of measured gene expression in each cluster attributed to mitochondrial gene (percent.MT) and the number of unique genes found for each nucleus (nFeature_RNA) were within expected values for each cell107.

The filtered data from HHT treated and control sample was analyzed using the standard integration protocol as implemented in Seurat v4.0.1108,109. This involved log normalization, variable feature selection and selecting/finding integration features/anchors. Cells were clustered using unsupervised Louvain clustering using the integrated nearest neighbors graph as input. The clustering resolution was determined empirically, as defined by the resolution with the greatest cluster stability (sc3_stability) from the clustree R package110,111. In the final dataset, there were 5342 and 6392 cells in the HHT and control conditions, with 14 total clusters, each present in both conditions. Cluster-specific markers were identified using the Wilcoxon Rank Sum test implemented in the FindAllMarkers function in Seurat. Lastly, differential gene expression between HHT and control was modeled using a hurdle MAST model112. We considered genes with an absolute fold change greater than 0.10 and Bonferroni-adjusted p value < 0.05 to be differentially expressed between conditions. These differentially expressed genes were then used as input for Gene Ontology (GO) enrichment analyses. Specifically, GO analysis was conducted for biological processes ontology with a minimum gene set size of 10 and maximum size of 500 using the clusterProfiler package113. For age-related analysis on snRNA-seq data, we performed additional analysis using SenMayo signature. SenMayo scores were calculated using Seurat’s ‘AddModuleScore’ function114, which computes the average expression levels of each gene set—in this case, the SenMayo set46—at the single-cell level. These scores are then normalized by subtracting an aggregated expression of control feature sets. To account for differences in total expression, all analyzed genes were first binned based on their average expression, and control features were then randomly selected from each bin. The SenMayo signature scores were subsequently compared between PBS- and HHT-treated groups using Wilcoxon’s rank sum test, with p values adjusted for multiple comparisons using FDR correction.

Drug affinity responsive target stability (DARTS)
The DARTS procedure was performed as previously described51. Briefly, cell lysates of RS HDFs were incubated with 50 μM HHT for 2 h at room temperature with rotation, and then 2 μl of the indicated protease solution was added to 20 μL samples of lysate. After incubation for 30 min at room temperature, digestion was stopped by adding a protease inhibitor cocktail. Proteins were separated by SDS-PAGE and observed with silver staining. Gel bands showing increased stability were cut out and prepared for mass spectrometry analysis to characterize proteins (Genomine Biotechnology, Pohang, Republic of Korea).

ATPase activity measurement
ATPase activity was measured by an enzyme-coupled spectrophotometric assay using ATPase Activity Assay Kit (Sigma-Aldrich, #MAK113) following the manufacturer’s instructions. Briefly, we established a standard curve using phosphate standards (#MAK113B). The reaction solution (40 mM Tris, 80 mM NaCl, 8 mM MgAc2, 1 mM EDTA (pH 7.5), and 4 mM ATP) was incubated with HSPA5 protein (5 μg) alone or HSPA5 protein and 10 μM inhibitor (HA15) at room temperature for 60 min. Each well was added with 200 µl of reagent (#MAK113A) and then incubated for 30 min at room temperature to terminate the enzyme reaction. The optical density of colorimetric product was measured using a microplate reader (TECAN, Männedorf, Switzerland) at 620 nm. The obtained absorbance was substituted into the standard curve to calculate the ATPase activity.

Surface plasmon resonance
Measurements of the apparent dissociation constants (KD) between HSPA5 and HHT were carried out using a Biacore T200 biosensor (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The recombinant human HSPA5 was bound covalently to the sensor chip CM5 (carboxylated dextran matrix) using an amine-coupling method. The human HSPA5 (20 μg/mL) in 10 mM sodium acetate pH 4.0 was coupled via injection, followed by the injection of 1 M ethanolamine to deactivate residual amines. For kinetic measurements at 20 °C, HHT with concentrations ranging from 1.5625 to 100 μM was prepared by dilution in HBS-P buffer (10 mM of HEPES (pH 7.4), 150 mM NaCl and 0.05% v/v surfactant P20 and 2% DMSO). Based on the results, the association rate constants (ka), dissociation rate constants (kd), and the equilibrium dissociation (binding) constants (KD; kd/ka) were determined.

Docking study
Possible binding model between HSPA5 and HHT was predicted by docking studies based on the X-ray structures of HSPA5 and its known inhibitor VER155008. The binding site of HHT is presumed to be like VER155008 and the related PDB structure (6CZ1.pdb) was referenced for docking study. The predicted binding model was verified considering the interaction between HSPA5 and VER155008. The initial binding model was determined by rigid docking study and MM-GBSA was performed for the final binding model prediction. MAESTRO (Schrödinger LLC, NY, USA) was used all calculational processes using Glide and Prime modules on Linux environment with default parameters115.

Mass cytometry (CyTOF) study and data processing
For CyTOF, stromal vascular fraction (SVF) and splenic immune cells were isolated from WAT and spleen, respectively. Visceral WAT and spleen are dissected, and finely minced. Adipose tissue was digested with Adipose tissue dissociation kit (Miltenyi Biotec, Gaithersburg, MD, USA) using gentleMACS™ Octo Dissociator with Heater (Miltenyi Biotec, Gaithersburg, MD, USA). Splenic cells were washed with PBS, neutralized with DMEM/F12 media supplemented with 10% FBS, and filtered using a 100 μm filter. WAT tissues were further digested with Type II collagenase (Worthington; #LS004176) in a 37 °C incubator for 30-40 min. Digested tissue was neutralized with DMEM/F12 media supplemented with 10% FBS and filtered using a 100 μm filter. The flow-through was centrifuged for 5 min at 400 x g. After removing the floating adipocyte fraction and supernatant, red blood cell lysis buffer (Sigma-Aldrich, #R7757) was added and incubated at room temperature for 2-3 min. The red blood cell lysis buffer is neutralized with DMEM/F12 media (supplemented with 10% FBS) and centrifuged for 5 min at 400 x g. The dissociated cells from adipose tissue or spleen were washed in a cell staining medium (CSM) and each sample was barcoded before staining according to the protocol described in the Cell-ID 20-Plex Pd Barcoding kit (Fluidigm, San Francisco, CA). Barcoded samples were pooled into 1 sample and stained with metal-tagged antibodies (Supplementary Table 8) as previously described67. Fixation and permeabilization were conducted according to the manufacturer’s instructions (Foxp3/Transcription Factor Staining Buffer Set, eBioscience). Finally, samples were washed and suspended in Maxpar Cell Acquisition Solution containing EQ normalization beads (Fluidigm, #201237) and data was collected by Helios instrument. FCS files were normalized using EQ beads by the Helios software. All data processing and analysis was done with Cytobank Enterprise (Beckman Coulter).

Statistical analysis
All statistical analyses were assessed by using Prism version 8 (Graph Pad Software Inc; La Jolla, CA, USA). The results are presented as the means ± standard error. In in vitro experiments, we conducted at least three independent experiments. Differences among the three or more groups were analyzed via one-way or two-way analysis of variance (ANOVA) followed by Tukey HSD test. Differences between two groups were analyzed by two-tailed Student’s t test. A p-value below 0.05 was considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors have made a commendable effort in addressing the previous reviewer concerns and added substantial new data. I have a few more comments which, if addressed, would further improve the manuscript for publication.\nThe study relies primarily on LDH and CCK-8 assays to test cytotoxicity of senescent cells. However, these methods are based on cellular metabolism, and senescent cells are known to have abnormal metabolic profiles that can skew such readouts. Please validate the cell viability/cytotoxicity data using metabolism-independent methods, such as nuclear staining for direct cell counting (DAPI/Hoechst) to corroborate findings.\nCommon senescence markers of p16 and p21 are not interchangeable in many cases as they may represent distinct senescent subtypes and have different biological consequences. The authors used only p16 to evaluate senescence in vitro while they focused on p21 analysis in vivo tissues. It is unclear whether HHT targets both populations equally or selectively. It would be helpful to provide data on both p16 and p21 expression in the models studied.\nThe results on macrophage M1/M2-like signatures appear inconsistent. While the authors provide some reasonable interpretations in the manuscript, another possibility is that a subset of macrophages may themselves become senescent, particularly in aged or metabolically stressed adipose tissue, and contribute to the confounding transcriptomic signals. It would be worthwhile to test whether senescent macrophages are present in these tissues and whether HHT treatment impacts this population.\nUnder 0% FBS conditions, cells may enter quiescence. Please check quiescent markers (e.g., p27KIP1, low Ki-67) to see if the control cells are simply quiescent as it would confound the interpretation of “selective killing”.\nThe strongest in vivo effects of HHT are seen in adipose tissue. Is this because HHT accumulates preferentially in fat, or that its target HSPA5 is expressed in adipose to a greater extent than other tissues? Biodistribution or PK/PD data would be informative.\nTo further support HSPA5 as the senotherapeutic target of HHT, the authors should provide data showing HSPA5 expression in senescent vs non-senescent cells, and across the major tissues affected. This would better support the proposed mechanism of action.\nHHT extends lifespan in Zmpste24-/- and aged mice - median lifespan, maximum lifespan, or both? And how much % increase?\nMinor point. Please define abbreviations (Ep, RP, SC, and BAT) in Figure 2C and the main text where they first appear. And please provide the PCR primer sequences used in the method section.", "reviewer_2": "In the revised manuscript “Homoharringtonine, a novel senotherapeutic, mitigates diet- and age-associated obesity and insulin resistance and extends the lifespan of mice”, Kim et al have indeed addressed the insufficient description of their experimental methods. However, in terms of experimental content, they still fail to convince me that they truly understand the molecular mechanism of how HHT eliminates senescent cells. Although the effects of drug treatment seem to be clear in animal experiments, the data and arguments are inconsistent and logically inadequate when authors investigate the impact of HHT on the immune microenvironment. These inconsistencies may suggest either instability in data quality or that the authors’ hypothesis regarding the senolytic effect of HHT is not entirely valid, thereby diminishing the clinical relevance of the study. However, if the focus is solely on HHT’s effects in a high-fat diet model, I believe the authors have made a considerable effort in this aspect.\n1. Regarding the authors’ response to Point 2, I now understand the reason for using 0% FBS. In both studies of ABT263 and D+Q, apoptosis is a critical indicator for evaluating senolytic efficacy, rather than changes in cell number. Therefore, in conclusion, I believe the authors are not required to perform experiments under 0% FBS conditions (for reasons to be explained below). However, they do need to clearly demonstrate whether the reduction in cell numbers for senescent and normal cells is due to cell cycle arrest or apoptosis. However, since 100 nM HHT reduced the number of NS HPAs and HDFs by approximately 40% and 25%, respectively, it is essential for the authors to describe how HHT passed the “toxicity to NS cells” screening threshold in Fig. S1I.\nAs the authors noted, low concentrations of FBS reduce cell proliferation, inducing a quiescent state and potentially promoting autophagy. While the authors intended to demonstrate that HHT was not toxic to normal cells undergoing cell cycle arrest, the observed difference could also result from autophagy-mediated drug resistance. Moreover, it remains unclear how HSPA5 expression and translational activity are affected under these conditions, which further confounds the explanation of the differential response between senescent and normal cells.\n2. As the authors mentioned, if HHT reduces p53 and p21 levels by inhibiting translation, this mechanism does not explain how HHT induces G1 cell cycle arrest in normal cells. Furthermore, it also fails to illustrate why p53 and p21 levels remain unchanged in 0% FBS-treated NS cells, as shown in Fig. 1K. In addition, the description of Fig. 1K was missing in the manuscript.\n3. Regarding the authors’ response to Point 6, they cited several references to argue that changes in the M1/M2 ratio within adipose tissue may not reliably reflect the inflammatory state. Through this reasoning, they attempt to rationalize the inconsistency between the increased M1/M2 ratio observed in the snRNA-seq data and the results from histological analyses. However, among the cited literature, one study explicitly states that although classical markers may not clearly distinguish M1 and M2 macrophages, the MacSpectrum characterization can still identify M1-like and M2-like phenotypes (PMID: 30990466). This study also confirmed that in obese mice, M1-like macrophages increase while M2-like macrophages decrease.\nIf the authors insist that neither classical markers nor transcriptomic signatures can indicate the inflammatory status of macrophages in adipose tissue, then why do they present a separate analysis of M1 and M2 populations in the CyTOF data shown in Fig. S15B, claiming a significant decrease in M1 macrophages? Is it possible that the authors separated M1 and M2 populations in the analysis because combining them would not have resulted in statistical significance?\nIn addition, the authors stated that “to precisely assess the inflammatory status of ATMs, they analyzed CLS using F4/80 staining.” However, as the authors mentioned, F4/80 is a pan-macrophage marker expressed by various macrophage subtypes. In fact, if expression level is to be considered, pro-inflammatory macrophages—such as M1 or certain monocyte-derived populations—may express even lower levels of F4/80 compared to M2 or tissue-resident macrophages, as noted in the references cited by the authors and others (PMID: 37651193). Furthermore, according to the CyTOF result, M1 macrophages constituted only a small fraction of the total macrophage population, which was strongly inconsistent with the F4/80 staining patterns observed in CLS. Because both CyTOF and snRNA-seq are relatively robust and unbiased techniques, I request that the authors provide whole-slide imaging data to allow for a more rigorous evaluation.\n4. Regarding the authors’ response to Point 7, I was asking the “Isotype control” but not “without primary antibody”.\n5. Regarding the authors’ response to Point 8, according to the data provided by the authors: (1) HSPA5 was highly expressed in cluster 8 and cluster 10, rather than in the APCs as the authors claimed. (2) The expression levels of HSPA5 in macrophages were comparable to that in APCs, but after HHT treatment, both HSPA5 and SenMayo scores significantly increased in macrophages. This is inconsistent with the authors’ claim that HHT eliminates HSPA5-expressing cells and reduces senescence markers. (3) If this senolytic effect is indeed cell type specific, then elucidating the molecular mechanism is critical for understanding their findings in vivo. (4) Although HSPA5 expression was reduced in APCs, SenMayo scores decreased in adipocytes, and the authors appear to conflate these two distinct observations.\nIn their response, the authors stated that HSPA5 expression positively correlates with SenMayo scores. I must reiterate that this claim is not supported by any data; specifically, the authors did not demonstrate that APCs with high SenMayo scores in the HF-PBS group expressed higher levels of HSPA5 compared to APCs with low SenMayo scores. The violin plots and bar graphs provided by the authors are insufficient to show their claim.\n6. Regarding the authors’ response to Point 9, based on the raw data provided by the authors, the absence of the siHSPA5 + Mock group makes it impossible to determine whether HSPA5 knockdown alone induces cell death. Moreover, the authors’ explanation does not clarify whether the survival of senescent cells is dependent on HSPA5 activity.\n7. In Fig. 8G, the SA-β-Gal signal appears to localize to structures like bronchi or blood vessels, with nearly all cells within the circular region showing positive staining. Is the author attempting to suggest that all cells in this region are senescent? Furthermore, does the spatial distribution of p53 signal in this same area within the tissue?", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "RESPONSE to REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have made a commendable effort in addressing the previous reviewer\nconcerns and added substantial new data. I have a few more comments which, if\naddressed, would further improve the manuscript for publication.\nOur response:\nWe sincerely thank the reviewer for the positive feedback and for recognizing our efforts\nto address the previous concerns with substantial new data. We also appreciate the\nadditional constructive comments provided. We carefully addressed each of these\npoints in the revised manuscript to further improve its clarity, rigor, and overall quality.\nThe study relies primarily on LDH and CCK-8 assays to test cytotoxicity of senescent\ncells. However, these methods are based on cellular metabolism, and senescent cells\nare known to have abnormal metabolic profiles that can skew such readouts. Please\nvalidate the cell viability/cytotoxicity data using metabolism-independent methods, such\nas nuclear staining for direct cell counting (DAPI/Hoechst) to corroborate findings.\nOur response:\nAccording to the reviewer’s suggestion, non-senescent (NS) and replicative senescent\n(RS) HDFs and HPAs were incubated in culture media containing 0% FBS for 1 day and\nsubsequently treated with the indicated concentrations of HHT for 4 days. After\ntreatment, cells were stained with SAG and eosin, and then cell numbers were\nquantified by directly counting cells from bright-field microscopic images using ImageJ\n(Figs. A–D, see below). This metabolism-independent approach yielded results\nconsistent with those obtained using the CCK-8 assay (Figs. 1I and S3K). Furthermore,\nthe concordance between CCK-8 assays and direct cell counting methods has been\nwell established in previous studies (PMID: 31460316).\n1\nas they may represent distinct senescent subtypes and have different biological\nconsequences. The authors used only p16 to evaluate senescence in vitro while they\nfocused on p21 analysis in vivo tissues. It is unclear whether HHT targets both\npopulations equally or selectively. It would be helpful to provide data on both p16 and\np21 expression in the models studied.\nOur response:\nWe thank the reviewer for raising this important point regarding the differential roles of\np16 and p21 as senescence markers. In the revised manuscript, we included p21 data\nin Supplementary Figure 1 (Fig. S1) of our in vitro analyses, showing that the\nexpression levels of p21 and p16 proteins were increased in senescent cells compared\nto non-senescent (NS) cells. For the human fat explant culture experiment shown in\nFigure 6N, we attempted to detect p16 by Western blotting but were unable to obtain\nreliable results, likely due to low protein abundance and technical limitations associated\nwith this tissue type. Given these constraints, we focused on presenting p53 and p21\nexpression data for the in vivo human samples, as both are well-established markers of\nsenescence and together provide robust evidence supporting our conclusions. In mouse\nsamples, we have already included the levels of p53, p21, and p16 proteins. Although\np16- and p21-positive cells may represent heterogeneous senescent subtypes, our data\nshow that both markers were reduced by HHT treatment in vitro and in vivo mouse\nmodels. These findings suggest that HHT exerts senotherapeutic effects across both\np16- and p21-defined populations.\nThe results on macrophage M1/M2-like signatures appear inconsistent. While the\nauthors provide some reasonable interpretations in the manuscript, another possibility is\nthat a subset of macrophages may themselves become senescent, particularly in aged\nor metabolically stressed adipose tissue, and contribute to the confounding\ntranscriptomic signals. It would be worthwhile to test whether senescent macrophages\nare present in these tissues and whether HHT treatment impacts this population.\nOur Response:\nWe thank the reviewer for this insightful comment. We agree that a subset of\nmacrophages may themselves become senescent, particularly in aged or metabolically\nstressed adipose tissue, and that this could contribute to the transcriptomic patterns\nobserved in our study. While our current study was not designed to directly identify\nsenescent macrophages, the increases in SenMayo and HSPA5 scores in\nmacrophages following HHT treatment suggest that HHT’s effects on this population\nmay differ from its impact on adipose cell lineages (Cluster 0, 1, 3, please see violin\nplots and bar graphs below). These findings support the concept of cell type–specific\n4\nresponses to HHT, in which macrophages may either undergo distinct senescence\npathways themselves, or experience heightened functional stress following the\nclearance of senescent adipose cells. The latter scenario suggests that, after the\nremoval of senescent or damaged adipose cells by HHT, surviving macrophages face a\n\"greater burden of debris clearance\", leading to increased functional stress and\naccelerated acquisition of senescent phenotypes (PMID: 38572110, 28721272).\nIn the revised manuscript, we expanded the Discussion and Limitations sections to\ninclude this interpretation, emphasizing that recognizing and understanding such cell\ntype–specific effects is critical for evaluating the broader applicability and limitations of\nHHT as a therapeutic agent. We also consider the identification and characterization of\nsenescent macrophages, as well as the assessment of their responses to HHT, to be an\nimportant focus for future studies.\nThese new sentences were added in the Discussion section.\n“Consistently, we found that HHT treatment reduced the number of adipose macrophages, as\nevidenced by a decrease in crown-like structure (CLS). However, despite the significant reduction\nin total macrophage numbers, HHT-treated macrophages exhibited higher levels of age-associated\ngene signature (e.g., SenMayo) and a stronger M1-like signature, which contrasted with the overall\nreduction in tissue inflammation. This discrepancy may partly reflect methodological differences\nbetween transcriptomic and protein-based analyses. Recent evidence indicates that macrophage\nactivation states can persist even after adipose inflammation resolves, due to epigenetic imprinting\nor stress adaptation79,80. Accordingly, we propose that following HHT-induced apoptosis of\nsenescent adipose cells, macrophages undergo transient stress responses associated with debris\nclearance, leading to pro-inflammatory and senescence-like features81,82. These findings suggest\nthat the observed signatures represent temporary adaptive states rather than persistent\ninflammation, highlighting the need for future studies to define cell type–specific responses to HHT.\nWe further speculate that these discrepancies may arise from differences in macrophage status or\ndisease conditions; for instance, in the published study83, HHT suppressed intestinal inflammatory\nsignatures when administered after inflammation had already been established, whereas in our\nstudy, mice were treated with HHT during the HF feeding period.”\nmarkers (e.g., p27KIP1, low Ki-67) to see if the control cells are simply quiescent as it\nwould confound the interpretation of “selective killing”.\nOur Response:\nIn accordance with the reviewer’s suggestion, we examined the expression of the\nproliferation marker Ki-67 in HPAs and HDFs cultured under 10% and 0% FBS\nconditions by Western blotting and immunofluorescence staining (Fig. A and B). Under\n0% FBS, Ki-67 expression was very low in HPAs and undetectable in HDFs, indicating\nthat the cells were in a quiescent state under these conditions. We acknowledged the\npotential quiescence of non-senescent cells, which may enable them to evade HHT-\ninduced cell death.\nWe added the following sentences in the Limitation section of the revised manuscript.\n\"HHT-induced cytotoxicity was not observed in NS cells, particularly under serum-free (0%\nFBS) conditions. This resistance may be attributed to serum starvation-induced quiescence,\nwhich could enable cells to evade HHT's cytotoxic effects.”\nThe strongest in vivo effects of HHT are seen in adipose tissue. Is this because HHT\naccumulates preferentially in fat, or that its target HSPA5 is expressed in adipose to a\ngreater extent than other tissues? Biodistribution or PK/PD data would be informative.\nOur response:\nA previous study demonstrated that HSPA5 (GPR78) is highly expressed in adipose\ntissue (especially visceral adipose tissue) and is elevated with aging, obesity and\ndiabetes in both humans and mice (please see below, adapted from PMID: 34615619).\nThese findings support why adipose tissue is a major target organ of HHT.\nday of treatment was 0.55 hours, and the mean Cmax was 25.1 ng/mL. However,\nPK/PD data for HHT in mice are currently unavailable.\nAforementioned study (PMID: 34615619) identified HSPA5 as a binding partner of the\nSARS-CoV-2 spike protein, facilitating its interaction with angiotensin-converting\nenzyme 2 (ACE2) and thereby increasing the risk of severe COVID-19 progression.\nConsistent with this, recent studies have shown that HHT can limit SARS-CoV-2\nprogression (PMID: 34676091, 34162861), potentially through its targeting of HSPA5.\nTogether, these findings highlight HHT’s potential to exert broad beneficial effects\nacross metabolic, infectious, and age-associated pathologies, with adipose tissue as a\nparticularly responsive site of action.\nIn summary, we are only at the early stages of understanding how HHT improves\nmetabolic health and mitigates age-associated disease. Further mechanistic\ninvestigations, in vivo biodistribution, and PK/PD analyses are warranted to fully\ndelineate its tissue-specific activity and translational potential.\nWe added the following sentences in the Limitation section of the revised manuscript.\n“We observed the most pronounced in vivo effects of HHT in adipose tissue. A plausible\nexplanation is that HSPA5 is highly expressed in adipose tissue, particularly in visceral adipose\ntissue, and its expression increases with aging, obesity, and diabetes in both humans and mice90.\nThese findings support the notion that adipose tissue may serve as a major target organ of HHT.\nAnother possibility is that HHT preferentially accumulates in adipose tissue compared to other\norgans. However, tissue distribution and pharmacological action of HHT in mice are currently\nunavailable. Therefore, future pharmacokinetic and pharmacodynamic studies are warranted to\ndetermine the tissue distribution of HHT and to clarify its primary target sites.”\nTo further support HSPA5 as the senotherapeutic target of HHT, the authors should\nprovide data showing HSPA5 expression in senescent vs non-senescent cells, and\nacross the major tissues affected. This would better support the proposed mechanism of\naction.\nOur response:\nIn response to the reviewer’s comment, we conducted additional experiments to further\ncharacterize HSPA5 expression. We found that HSPA5 protein levels were higher in\nsenescent cells than in non-senescent (NS) cells across HPAs, HDFs, and hRPEs (Fig.\nS11E, please see below). Moreover, we examined HSPA5 expression in other major\nmetabolic tissues, including skeletal muscle (tibialis anterior) and liver, from chow-fed\nand high-fat diet (HF)-fed mice. HSPA5 protein levels were significantly increased in\nboth adipose tissue and skeletal muscle of HF-fed mice compared with chow-fed\ncontrols, whereas its expression remained largely unchanged in the liver (Fig. 7N and\nS11J). The more pronounced increase in HSPA5 expression within adipose tissue\nsuggests that adipose tissue is a primary metabolic target organ responsive to HHT\ntreatment.\nFig. S11J (New data)\nWe added the following sentence in the Result section of the revised manuscript.\n\"HSPA5 protein levels were consistently higher in senescent cells than in NS cells across HPAs,\nHDFs, and hRPEs (Fig. S11E).\"\n“In metabolic tissues, HSPA5 protein levels were significantly increased in both adipose tissue and\nskeletal muscle from HF-induced obese mice compared with chow-fed controls, whereas its\nexpression was not significantly changed in the liver (Figs. 7N and S11J). The increase in HSPA5\nexpression was more prominent in adipose tissue than in skeletal muscle, suggesting that adipose\ntissue is a primary metabolic target organ responsive to HHT treatment.”\nTo further investigate whether HSPA5 serves as a senotherapeutic target of HHT, both\nNS and RS HPAs were treated with 100 nM HHT for 2 days—a time window selected to\ncapture the induction and progression phase of apoptosis without inducing overt cell\ndeath—and subsequently subjected to co-immunostaining for HSPA5 and cleaved\ncaspase-3 (Casp-3). HSPA5 was detected using a horse radish peroxidase (HRP)-\nconjugated anti-mouse secondary antibody with 3,3'-diaminobenzidine (DAB)\nchromogen, whereas Casp-3 was visualized using an alkaline phosphatase (AP)-\nconjugated anti-rabbit secondary antibody with Fast Red chromogen. Consistently, RS\nHPAs displayed higher HSPA5 and Casp-3 levels than NS HPAs (Fig. 7G). Notably,\namong the HHT-treated RS HPAs, cells with high HSPA5 expression exhibited a\nsignificantly greater number of Casp-3-positive cells compared to those with low HSPA5\nexpression (Fig. 7H). Furthermore, HHT treatment in HF-fed obese mice led to reduced\nto the selective elimination of HSPA5 high-expressing senescent cells, as confirmed by\nimmunohistochemistry (Fig. 7O). Collectively, these additional data strengthen our\nmechanistic insight and support the conclusion that HSPA5 might act as a key mediator\nof the senotherapeutic activity of HHT in HPAs.\nFig. 7G, 7H, and 7O (New data)\nWe added the following sentences in the Result section of the revised manuscript.\n“To further confirm whether the senotherapeutic activity of HHT might be mediated through\nHSPA5, NS and RS HPAs were treated with 100 nM HHT for 2 days–a time window selected to\ncapture the induction and progression of apoptosis without causing overt cell death–and\nsubsequently subjected to co-immunostaining for HSPA5 and Casp-3, using DAB (brown) and\nFast Red (red) as chromogens, respectively. RS HPAs treated with HHT consistently showed\nhigher expression of HSPA5 and Casp-3 than HHT-treated NS HPAs (Fig. 7G). Among HHT-\ntreated RS HPAs, cells with high HSPA5 expression showed a significantly greater number of\nCasp-3-positive cells compared with those expressing low HSPA5 (Fig. 7H). These data suggest\nthat HSPA5 high expressing cells are primary target of HHT, and HSPA5 may serve as a key\nmediator of the senotherapeutic activity of HHT.”\nlifespan, or both? And how much % increase?\nOur response:\nAlthough Figures 8A, 9B, and 9G already present these data, in accordance with the\nreviewer’s suggestion, we additionally calculated the median and maximum lifespans. In\nboth male and female Zmpste24-/- mice (Fig. 8A), HHT treatment extended the median\nlifespan from 14 to 15 weeks and the maximum lifespan from 15 to 19 weeks,\ncorresponding to a 7.14% increase in median lifespan and a 26.67% increase in\nmaximum lifespan. In aged mice (Fig. 9B), the median lifespan of 24 months could be\ndetermined only in the PBS-treated group. This is because, after 50% of the PBS-\ntreated mice had died and a statistically significant difference in survival was observed\nbetween the PBS and HHT groups, the remaining animals were sacrificed for blood,\nurine, and tissue analyses rather than being followed until natural death. In an additional\ncohort (Fig. 9G), the median lifespan was 509 days in the PBS group and 551 days in\nthe HHT group, corresponding to an 8.25% increase following HHT treatment. However,\nthe maximum lifespan could not be determined because the mice were sacrificed at 558\ndays for blood, urine, and tissue analyses.\nWe included the followings in the Results section of the revised manuscript.\n“HHT treatment significantly extended lifespan, increasing the median lifespan from 14 to 15 weeks\nand the maximum lifespan from 15 to 19 weeks (Fig. 8A).”\n“We found that HHT significantly extended the lifespan of mice, increasing the median lifespan\nfrom 509 to 551 days, whereas the maximum lifespan could not be determined because the mice\nwere sacrificed at 558 days for tissue collection (Fig. 9G).”\nMinor point. Please define abbreviations (Ep, RP, SC, and BAT) in Figure 2C and the\nmain text where they first appear. And please provide the PCR primer sequences used\nin the method section.\nOur response:\nWe defined the above-mentioned abbreviations in the Figure legend of Fig. 2. We have\nalready included the PCR primer sequences in Supplementary Table 9.\nIn the revised manuscript “Homoharringtonine, a novel senotherapeutic, mitigates diet-\nand age-associated obesity and insulin resistance and extends the lifespan of mice”,\nKim et al have indeed addressed the insufficient description of their experimental\nmethods. However, in terms of experimental content, they still fail to convince me that\nthey truly understand the molecular mechanism of how HHT eliminates senescent cells.\nAlthough the effects of drug treatment seem to be clear in animal experiments, the data\nand arguments are inconsistent and logically inadequate when authors investigate the\nimpact of HHT on the immune microenvironment. These inconsistencies may suggest\neither instability in data quality or that the authors’ hypothesis regarding the senolytic\neffect of HHT is not entirely valid, thereby diminishing the clinical relevance of the study.\nHowever, if the focus is solely on HHT’s effects in a high-fat diet model, I believe the\nauthors have made a considerable effort in this aspect.\nOur response:\nWe thank the reviewer for this constructive and thoughtful comment. As correctly noted,\nour primary focus in this study is to investigate the effects of HHT in the high-fat diet\n(HFD) model, where we observed robust improvements in adipose tissue inflammation,\ninsulin resistance, and overall metabolic health. Although we have also observed similar\neffects in human adipose tissue ex vivo, we fully agree that the clinical relevance of\nHHT requires future validation in well-designed translational studies.\nWe acknowledge the reviewer’s concern regarding certain inconsistencies in our\nmacrophage data. In this revised version, we have carefully reanalyzed these datasets\nand provided detailed explanations of the possible biological reasons underlying the\nobserved variability (e.g., differences in tissue context, treatment duration, or cell-type–\nspecific responses).\nWe believe these clarifications now offer a more coherent interpretation of how HHT\nmodulates immune (i.e., macrophages) and senescent cell interactions. We sincerely\nappreciate the reviewer’s insightful feedback, which has significantly improved the\nclarity and depth of our manuscript. We hope that the revisions adequately address the\nreviewer’s concerns and convey a more complete understanding of HHT’s\nsenotherapeutic actions in metabolism- and age-associated pathological conditions.\n1. Regarding the authors’ response to Point 2, I now understand the reason for using\n0% FBS. In both studies of ABT263 and D+Q, apoptosis is a critical indicator for\nevaluating senolytic efficacy, rather than changes in cell number. Therefore, in\nconclusion, I believe the authors are not required to perform experiments under 0% FBS\nconditions (for reasons to be explained below). However, they do need to clearly\ndemonstrate whether the reduction in cell numbers for senescent and normal cells is\ndue to cell cycle arrest or apoptosis. However, since 100 nM HHT reduced the number\n12\nof NS HPAs and HDFs by approximately 40% and 25%, respectively, it is essential for\nthe authors to describe how HHT passed the “toxicity to NS cells” screening threshold in\nFig. S1I.\nOur response:\nUnder both 0% and 10% FBS conditions, the senolytic effects of HHT on RS HPAs and\nH\nand\nHDFs were mediated by apoptosis, as evidenced by increased cleaved caspase-3 and\ndecreased PARP expression in Western blot analyses (Figs. 1K, S3D, S3H, and S3M).\nConsistently, LDH activity in the culture media was elevated in these cells (Figs. 1J,\nS3C, S3G, and S3L). In contrast, non-senescent cells showed no detectable cleaved\ncaspase-3, no change in PARP expression, and no increase in LDH activity, indicating\nthe absence of apoptosis (Figs. 1J, 1K, S3C, S3D, S3G, S3H, S3L, and S3M).\nFurthermore, flow cytometry demonstrated that HHT treatment induced cell cycle arrest\nin non-senescent cells under 10% FBS conditions (Fig. S3I). Collectively, these findings\nindicate that HHT selectively induces apoptosis in senescent cells, whereas it primarily\ncauses cell cycle arrest in non-senescent cells.\nWe would like to clarify that HPAs were not included in the initial screening processes\nfor toxicity and senolytic assays. In the case of HDFs, treatment with HHT (#34 in the\nTable below) showed 45.19 ± 1.35% cell survival in RS HDFs and 96.59 ± 2.42% in NS\nHDFs during the initial screening. Therefore, HHT passed the “toxicity to NS cells”\nthreshold, which we defined as ≥75% cell survival. Although the data in Fig. S3F\n(previously Fig. S1I) indicate that HHT treatment resulted in approximately a 25%\nreduction in cell number for NS HDFs, we considered this acceptable because it still\nmeets our predefined criterion for toxicity. We believe that the apparent discrepancy in\ntoxicity between the initial screening and the subsequent experiment is due to\ndifferences in the cellular state, which likely influence the sensitivity of NS HDFs to HHT\ntreatment.\n13\nquiescent state and potentially promoting autophagy. While the authors intended to\ndemonstrate that HHT was not toxic to normal cells undergoing cell cycle arrest, the\nobserved difference could also result from autophagy-mediated drug resistance.\nMoreover, it remains unclear how HSPA5 expression and translational activity are\naffected under these conditions, which further confounds the explanation of the\ndifferential response between senescent and normal cells.\nOur response:\nWe appreciate this insightful comment. HHT-induced cytotoxicity was not observed in\nnon-senescent (NS) cells, particularly under 0% FBS conditions, which likely reflects\nserum starvation-induced quiescence rather than inherent resistance of normal cells to\nreduced metabolic activity and cell cycle arrest. In fact, the Reviewer 1 also asked a\nsimilar question and suggested to test quiescent markers (e.g., p27KIP1 or low Ki-67).\nUnder 0% FBS, Ki-67 expression was very low in HPAs and undetectable in HDFs,\nindicating that the cells were in a quiescent state under these conditions (Figure A and\nB, please see below). Additionally, we cannot exclude the possibility that autophagy\nactivation under serum-depleted conditions contributed to drug resistance, as\nautophagy is known to serve as a cytoprotective mechanism against various cellular\nstresses, including chemotherapeutic agents. We acknowledged the potential\nquiescence and autophagy activation of non-senescent cells, which may enable them to\nevade HHT-induced cell death, as a limitation of our study.\nWe included the following sentence in the Limitation section of the revised manuscript.\n\"HHT-induced cytotoxicity was not observed in non-senescent (NS) cells, particularly under\nserum-free (0% FBS) conditions. This resistance may be attributed to serum starvation-induced\nquiescence, which could enable cells to evade HHT's cytotoxic effects. Additionally, autophagy\nactivation under serum-depleted conditions may have contributed to drug resistance, as autophagy\nfunctions as a well-established cytoprotective mechanism against various cellular stresses,\nincluding chemotherapeutic agents.\"\nWe appreciate the reviewer’s insightful comment regarding the potential influence of\nserum deprivation on HSPA5 expression and translational activity. We acknowledge that\nlow-serum conditions may alter HSPA5 levels or induce autophagy-related adaptive\nresponses, which could in theory contribute to differential drug sensitivity. However,\nthese aspects were beyond the primary scope of our current study because the vast\nmajority of our experiments, including those assessing HSPA5 expression, protein\nsynthesis, and cellular responses to HHT, were conducted under standard culture\n16\nconditions with 10% FBS, where cells maintain normal proliferative and translational\nprofiles. The serum-free condition (0% FBS) was employed only as a transient\nquiescent control to demonstrate that HHT is not cytotoxic to non-senescent cells\narrested in the cell cycle. Thus, our key conclusions are derived from physiologically\nrelevant serum conditions rather than from serum-deprived states. We fully agree that\nfuture studies should examine how serum deprivation or autophagy induction may\nmodulate HSPA5 expression and contribute to drug resistance mechanisms. Such\ninvestigations will further clarify whether autophagy-mediated adaptations influence\nHHT sensitivity in different cellular contexts.\n2. As the authors mentioned, if HHT reduces p53 and p21 levels by inhibiting\ntranslation, this mechanism does not explain how HHT induces G1 cell cycle arrest in\nnormal cells. Furthermore, it also fails to illustrate why p53 and p21 levels remain\nunchanged in 0% FBS-treated NS cells, as shown in Fig. 1K. In addition, the description\nof Fig. 1K was missing in the manuscript.\nOur response:\nIn the current study, we did not investigate the specific mechanism underlying HHT-\ninduced G1 arrest in normal cells. Given that HHT is a well-established inhibitor of\nprotein translation in actively proliferating cells (PMID: 30975912), it is plausible that it\nreduces the synthesis of key cell-cycle regulators, thereby contributing to G1 arrest.\nRegarding the unchanged p53 and p21 levels in 0% FBS-treated NS cells (Fig. 1K), our\ntemporal analysis showed that both proteins decreased during the first day of HHT\ntreatment and gradually returned to baseline by day 4. This transient change likely\nreflects an adaptive or compensatory response of NS cells to sustained translation\ninhibition, potentially via feedback mechanisms that restore cell-cycle checkpoint\ncontrol. We included these new temporal data in our rebuttal.\nWe have already mentioned about Fig. 1K in the main text and the Figure legend of the\noriginal manuscript as follows:\n17\n“Under these conditions, HHT induced significant cytotoxicity in senescent HPAs and HDFs while\nhaving no effect on NS cell numbers (Figs. 1H-K and S3J-M).” – manuscript page 6\n“K. Expression levels of age- or apoptosis-associated proteins (n=3 in each group).” – manuscript\npage 54\n3. Regarding the authors’ response to Point 6, they cited several references to argue\nthat changes in the M1/M2 ratio within adipose tissue may not reliably reflect the\ninflammatory state. Through this reasoning, they attempt to rationalize the\ninconsistency between the increased M1/M2 ratio observed in the snRNA-seq data and the results\nfrom histological analyses. However, among the cited literature, one study explicitly\nstates that although classical markers may not clearly distinguish M1 and M2\nmacrophages, the MacSpectrum characterization can still identify M1-like and M2-like\nphenotypes (PMID: 30990466). This study also confirmed that in obese mice, M1-like\nmacrophages increase while M2-like macrophages decrease.\nIf the authors insist that neither classical markers nor transcriptomic signatures can\nindicate the inflammatory status of macrophages in adipose tissue, then why do they\npresent a separate analysis of M1 and M2 populations in the CyTOF data shown in Fig.\nS15B, claiming a significant decrease in M1 macrophages? Is it possible that the\nauthors separated M1 and M2 populations in the analysis because combining them\nwould not have resulted in statistical significance? In addition, the authors stated that “to\nprecisely assess the inflammatory status of ATMs, they analyzed CLS using F4/80\nstaining.” However, as the authors mentioned, F4/80 is a pan-macrophage marker\nexpressed by various macrophage subtypes. In fact, if expression level is to be\nconsidered, pro-inflammatory macrophages—such as M1 or certain monocyte-derived\npopulations—may express even lower levels of F4/80 compared to M2 or tissue-\nresident macrophages, as noted in the references cited by the authors and others\n(PMID: 37651193). Furthermore, according to the CyTOF result, M1 macrophages\nconstituted only a small fraction of the total macrophage population, which was strongly\ninconsistent with the F4/80 staining patterns observed in CLS. Because both CyTOF\nand snRNA-seq are relatively robust and unbiased techniques, I request that the\nauthors provide whole-slide imaging data to allow for a more rigorous evaluation.\nOur response:\nWe thank the reviewer for these detailed comments and the opportunity to clarify this\npoint. We believe there may have been a misunderstanding regarding our analyses: we\ndid not use the “ratio” of M1- to M2-like macrophages in any of our data or in our\nprevious response. We fully acknowledge that computational characterization using\nMacSpectrum can distinguish M1-like and M2-like phenotypes. Indeed, this was the\nreference (PMID: 30990466) we relied upon for our macrophage analyses (Fig. S8D)\nand in our response to Point 6.\nWe wish to emphasize that the decision to present M1 and M2 populations separately in\nFig. S15B was not driven by statistical considerations. Rather, this analysis was\n18\nperformed in response to one of the reviewer’s earlier suggestions and aligns with our\noverall strategy of employing multiple complementary approaches—including Western\nblotting of NF-κB pathway components, cytokine profiling, CyTOF, and\nimmunohistochemical staining—to comprehensively assess inflammatory status.\nRegarding the reviewer’s comment on F4/80 staining, we have now included\nimmunofluorescence images using the M1-specific marker iNOS, which more directly\nreflects pro-inflammatory macrophages, instead of the F4/80-positive crown-like\nstructures (Fig. S8E).\nWe revised the following sentences in the Result section of the revised manuscript.\n“Despite the seemingly contradictory transcriptomic signatures, our immunofluorescent staining\nwith the M1-like macrophage marker iNOS further confirmed that CLS with iNOS-positive pro-\ninflammatory macrophages was significantly reduced in HHT-treated WAT (Fig. S8E).”\nFig. S8E (New data)\nTo further strengthen our response and address the reviewer’s request for greater rigor,\nwe have also provided whole-slide imaging data of F4/80 staining in this rebuttal (please\nsee below). We believe that these data, together with our multi-assay approach, offer a\nmore comprehensive and reliable evaluation of macrophage-associated inflammation in\nHHT-treated adipose tissue (please see below).\n19\ninconsistency between M1/M2-like signatures and the systemic metabolic\nimprovements with reduced adipose inflammation observed in HHT-treated mice. This\ndiscrepancy may in part reflect methodological differences between protein-based and\ntranscriptomic approaches.\nIn addition, recent studies have shown that obesity-induced macrophage activation\nsignatures may persist even after macrophage infiltration and adipose inflammation are\nresolved, likely due to epigenetic imprinting of adipose immune cells (PMID: 40634602,\n35618862).\nOur interpretation is that, following the removal of senescent or damaged adipose cells\nby HHT, adipose macrophages may experience an increased debris clearance burden,\nresulting in functional stress and accelerated acquisition of pro-inflammatory and\nsenescence-like features (PMID: 38572110, 28721272), which may persist even after\nadipose inflammation are resolved,\nCollectively, we speculate that the increased pro-inflammatory and senescence-\nassociated transcriptome signatures observed in HHT-treated macrophages reflect\npersistent inflammatory programs initiated during prior HHT-induced adipose cell death\n(PMID: 40634602). These sustained programs may underlie the heterogeneity and\napparent contradictions in our transcriptomic dataset.\nIn the revised manuscript, we have added new sentences in the Discussion section to\ninclude this interpretation and highlight the need for future studies to determine cell\ntype–specific effects of HHT, particularly in macrophages.\n“Consistently, we found that HHT treatment reduced the number of adipose macrophages, as\nevidenced by a decrease in crown-like structure (CLS). However, despite the significant reduction\nin total macrophage numbers, HHT-treated macrophages exhibited higher levels of age-associated\ngene signature (e.g., SenMayo) and a stronger M1-like signature, which contrasted with the overall\nreduction in tissue inflammation. This discrepancy may partly reflect methodological differences\nbetween transcriptomic and protein-based analyses. Recent evidence indicates that macrophage\nactivation states can persist even after adipose inflammation resolves, due to epigenetic imprinting\nor stress adaptation79,80. Accordingly, we propose that following HHT-induced apoptosis of\nsenescent adipose cells, macrophages undergo transient stress responses associated with debris\nclearance, leading to pro-inflammatory and senescence-like features81,82. These findings suggest\nthat the observed signatures represent temporary adaptive states rather than persistent\ninflammation, highlighting the need for future studies to define cell type–specific responses to HHT.\nWe further speculate that these discrepancies may arise from differences in macrophage status or\ndisease conditions; for instance, in the published study83, HHT suppressed intestinal inflammatory\nsignatures when administered after inflammation had already been established, whereas in our\nstudy, mice were treated with HHT during the HF feeding period.”\n21\n4. Regarding the authors’ response to Point 7, I was asking the “Isotype control” but not\n“without primary antibody”.\nOur response:\nWe now provide the isotype control data for the reviewer.\n5. Regarding the authors’ response to Point 8, according to the data provided by the\nauthors: (1) HSPA5 was highly expressed in cluster 8 and cluster 10, rather than in the\nAPCs as the authors claimed. (2) The expression levels of HSPA5 in macrophages\nwere comparable to that in APCs, but after HHT treatment, both HSPA5 and SenMayo\nscores significantly increased in macrophages. This is inconsistent with the authors’\nclaim that HHT eliminates HSPA5-expressing cells and reduces senescence markers.\n(3) If this senolytic effect is indeed cell type specific, then elucidating the molecular\nmechanism is critical for understanding their findings in vivo. (4) Although HSPA5\nexpression was reduced in APCs, SenMayo scores decreased in adipocytes, and the\nauthors appear to conflate these two distinct observations.\nOur responses:\nWe appreciate the reviewer’s careful attention to these points and the opportunity to\nclarify our interpretations.\nRegarding comment (1).\nWe acknowledge that clusters 8 and 10 express higher levels of HSPA5 compared to\nother cell types. In our dataset, cluster 8 shows higher expression of both SenMayo and\nHSPA5 than the APC population (cluster 2). Recent studies have identified mesothelial-\nlike cells (e.g., cluster 8) within rodent visceral adipose tissue (such as perigonadal fat).\nThese cells exhibit properties similar to APCs and have been implicated in adipose\ntissue fibrosis (PMID: 37886999, 36889280, 26412153). We believe that HHT may act\non mesothelial-like cells in a manner similar to its effects on APCs, as we observed a\ntrend toward reduced SenMayo and HSPA5 expression in cluster 8 following HHT\ntreatment. Given that these mesothelial-like cells and cluster 10 remain poorly\ncharacterized minor populations, we chose to focus primarily on the well-defined APC\npopulation (cluster 2) in the main discussion, but we do not exclude the possibility that\nHHT also impacts other cell types, including clusters 8 and 10.\nOur data suggest that HHT exerts a senolytic effect on certain adipose cell lineages,\nincluding APCs and adipocytes, but its effect on macrophages appears distinct.\nSpecifically, HHT-treated macrophages displayed increased HSPA5 and SenMayo\nscores, suggesting that HHT does not have senolytic activity in this cell type and may\ne\nmay\neven enhance senescence-like features. One plausible explanation is that, after the\nremoval of senescent or damaged adipose cells, surviving macrophages experience a\ngreater “debris clearance” burden during the early phase of HHT treatment, leading to\nincreased functional stress and accelerated acquisition of senescence phenotypes\n(PMID: 38572110, 28721272).\nHHT is known to act through multiple mechanisms, including inhibition of translation via\nbinding to the A-site cleft of the 60S ribosomal subunit (PMID: 24501394), depletion of\nshort-lived survival or oncogenic proteins such as MCL-1 (PMID: 20971952), induction\nof cell-cycle arrest at various phases (PMID: 33716735, 33548212), and inhibition of\nSTAT3 signaling (PMID: 26166037). HHT has also been reported to alleviate\nmacrophage-mediated inflammation through NF-κB pathway inhibition (PMID:\n36211988). However, direct evidence for HHT-mediated promotion of M1 macrophage\nactivation or increased inflammatory signaling is limited. Our findings suggest that HHT\nmay exert cell-type–specific effects, with macrophage responses potentially shaped\nmore by microenvironmental demands than by direct drug action. Future studies are\nwarranted to elucidate these differential mechanisms.\nAs already mentioned earlier, we incorporate this interpretation into the revised\nDiscussion section as follows to highlight the possibility of cell type–specific responses\nto HHT or HHT-treated microenvironmental influence on macrophages.\n“Consistently, we found that HHT treatment reduced the number of adipose macrophages, as\nevidenced by a decrease in crown-like structure (CLS). However, despite the significant reduction\nin total macrophage numbers, HHT-treated macrophages exhibited higher levels of age-associated\ngene signature (e.g., SenMayo) and a stronger M1-like signature, which contrasted with the overall\nreduction in tissue inflammation. This discrepancy may partly reflect methodological differences\nbetween transcriptomic and protein-based analyses. Recent evidence indicates that macrophage\nactivation states can persist even after adipose inflammation resolves, due to epigenetic imprinting\nor stress adaptation79,80. Accordingly, we propose that following HHT-induced apoptosis of\nsenescent adipose cells, macrophages undergo transient stress responses associated with debris\nclearance, leading to pro-inflammatory and senescence-like features81,82. These findings suggest\nthat the observed signatures represent temporary adaptive states rather than persistent\ninflammation, highlighting the need for future studies to define cell type–specific responses to HHT.\nWe further speculate that these discrepancies may arise from differences in macrophage status or\ndisease conditions; for instance, in the published study83, HHT suppressed intestinal inflammatory\nsignatures when administered after inflammation had already been established, whereas in our\nstudy, mice were treated with HHT during the HF feeding period.”\nWe appreciate the opportunity to clarify this point. As the reviewer pointed out, although\nHSPA5 expression was reduced in APCs, SenMayo scores were decreased in\nadipocytes. It has been shown that young and healthy APCs under high-fat diet\nconditions can differentiate into mature adipocytes (PMID: 34562641, 22596050). In line\nwith this, we observed an increased number of adipocytes in the HHT-treated group.\nBased on these findings, we speculate that healthy, young APCs with lower HSPA5\nexpression may have differentiated into new mature adipocytes, leading to an increase\nin newly formed adipocytes (Fig. 4A, 4B). This process, known as hyperplastic\nadipogenesis, represents a form of healthy adipose tissue remodeling (PMID:\n31573549, 37181756) and may contribute to the observed reduction in SenMayo scores\nin adipocytes.\nOur interpretation of cellular aging and inflammation does not rely on a single parameter\nor transcriptomic signature. Instead, we integrated multiple complementary assays—\nincluding protein-level analyses (NF-κB, inflammatory cytokines), CyTOF, and\nhistological evaluation, which consistently demonstrated reduced inflammatory\nsignaling, decreased M1 macrophage abundance, and reduced senescence markers in\nadipose tissue following HHT treatment. While some transcriptomic signatures (e.g.,\nSenMayo in macrophages) diverged from these protein-based observations, such\ndifferences are not unexpected and may reflect distinct biological contexts, assay\nsensitivities, or temporal dynamics.\nIn their response, the authors stated that HSPA5 expression positively correlates with\nSenMayo scores. I must reiterate that this claim is not supported by any data;\nspecifically, the authors did not demonstrate that APCs with high SenMayo scores in the\nHF-PBS group expressed higher levels of HSPA5 compared to APCs with low SenMayo\nscores. The violin plots and bar graphs provided by the authors are insufficient to show\ntheir claim.\nOur response:\nOur statement that “Hspa5 expression positively correlates with SenMayo scores” was\nbased on the observation that cell clusters with higher average SenMayo scores (e.g.,\nClusters 2, 8, and 10, please see below) also displayed higher average Hspa5\nexpression. This pattern was evident in our violin plots and bar graphs and was\nintended to highlight a cluster-level trend, rather than a cell-by-cell correlation within a\ngiven cluster. We agree with the reviewer that our current data do not directly\ndemonstrate that, within the APC population of the HF-PBS group, cells with high\nSenMayo scores express higher Hspa5 levels than those with low scores. Nonetheless,\nwe believe this cluster-level association reflects a biologically meaningful relationship\nbetween ER stress and cellular senescence, which we plan to examine in greater detail\nthrough future single-cell correlation analyses.\n24\n6. Regarding the authors’ response to Point 9, based on the raw data provided by the\nauthors, the absence of the siHSPA5 + Mock group makes it impossible to determine\nwhether HSPA5 knockdown alone induces cell death. Moreover, the authors’\nexplanation does not clarify whether the survival of senescent cells is dependent on\nHSPA5 activity.\nOur responses:\nWe agree that including the siHSPA5+Mock group is important to properly assess the\neffectiveness of HHT in the absence of HSPA5. As shown in Figure A, no significant\ndifference in cell number was observed between the scrambled siRNA (siSc)+Mock and\nsiHSPA5+Mock groups. For this reason, we used the siSC+Mock group as the control\nin the experiments presented in Figs. 7C–E. We hope this clarification addresses the\nreviewer’s concern regarding our interpretation of HHT’s mechanism of action. If\nnecessary, we can note this point in the Limitations section of the revised manuscript.\nRegarding whether the survival of senescent cells depends on HSPA5 activity, previous\nstudies have demonstrated that complete knockout of Grp78 (Hspa5) in mice causes\nembryonic lethality due to apoptosis of the inner cell mass (PMID: 16847323), and\nconditional knockout in the hematopoietic compartment results in cell death and\nimpaired oncogenic signaling (PMID: 21937694). These findings establish HSPA5 as an\nessential endoplasmic-reticulum chaperone required for cell survival, whose complete\ndisruption leads to severe cellular defects. Notably, partial depletion of HSPA5 by siRNA\n25\nor shRNA alters cellular plasticity and increases susceptibility to stress-induced cell\ndeath (PMID: 21937694, 39472623, 28130223). Collectively, these studies indicate that\nloss of HSPA5 can trigger cell death, although the outcome depends on cell type,\nknockdown efficiency, degree of senescence, and cellular stress context.\nMoreover, to clarify whether the survival of senescent cells depends on HSPA5 activity,\nwe performed additional experiment. In this new experiment, HPAs were transfected\nwith higher concentration of siHSPA5 (25 nM). After 4 days of incubation, cells were co-\ntreated with 500 nM doxorubicin (Dox) to enhance senescence induction and 100 nM\nHHT or PBS. Under this condition, the viability of siHSPA5+Mock cells was reduced to\n81.07 ± 2.54% relative to siSc+Mock cells (Figure B), indicating that survival of\nsenescent cells partially depends on HSPA5. Importantly, cell viability markedly\ndecreased to 43.61 ± 1.44% in siSc+HHT cells but remained high (81.94 ± 2.15%) in\nsiHSPA5+HHT cells, suggesting that a certain level of HSPA5 expression is required for\nthe senolytic activity of HHT. Notably, there was no significant difference in cell viability\nbetween siHSPA5+Mock and siHSPA5+HHT groups. Collectively, these findings\ndemonstrate that the senotherapeutic effect of HHT is strongly dependent on the\npresence of HSPA5.\nTo further investigate whether HSPA5 serves as a senotherapeutic target of HHT, both\nNS and RS HPAs were treated with 100 nM HHT for 2 days—a time window selected to\ncapture the induction and progression phase of apoptosis without inducing overt cell\ndeath—and subsequently subjected to co-immunostaining for HSPA5 and cleaved\ncaspase-3 (Casp-3). HSPA5 was detected using a horse radish peroxidase (HRP)-\nconjugated anti-mouse secondary antibody with 3,3'-diaminobenzidine (DAB)\nchromogen, whereas Casp-3 was visualized using an alkaline phosphatase (AP)-\nconjugated anti-rabbit secondary antibody with Fast Red chromogen.\nConsistently, RS HPAs displayed higher HSPA5 and Casp-3 levels than NS HPAs (Fig.\n7G). Notably, among the HHT-treated RS HPAs, cells with high HSPA5 expression\nexhibited a significantly greater number of Casp-3-positive cells compared to those with\nlow HSPA5 expression (Fig. 7H). Furthermore, HHT treatment in HF-fed obese mice led\nto reduced HSPA5 protein levels in adipose tissues compared with PBS-treated\ncontrols, likely due to the selective elimination of HSPA5 high-expressing senescent\n26\ncells, as confirmed by immunohistochemistry (Fig. 7O). Collectively, these additional\ndata strengthen our mechanistic insight and support the conclusion that HSPA5 might\nact as a key mediator of the senotherapeutic activity of HHT in HPAs.\nFig. 7G, 7H, and 7O (New data)\nWe added the following sentences in the Result section of the revised manuscript.\n“To further confirm whether the senotherapeutic activity of HHT might be mediated through\nHSPA5, NS and RS HPAs were treated with 100 nM HHT for 2 days–a time window selected to\ncapture the induction and progression of apoptosis without causing overt cell death–and\nsubsequently subjected to co-immunostaining for HSPA5 and Casp-3, using DAB (brown) and\nFast Red (red) as chromogens, respectively. RS HPAs treated with HHT consistently showed\nhigher expression of HSPA5 and Casp-3 than HHT-treated NS HPAs (Fig. 7G). Among HHT-\ntreated RS HPAs, cells with high HSPA5 expression showed a significantly greater number of\nCasp-3-positive cells compared with those expressing low HSPA5 (Fig. 7H). These data suggest\nthat HSPA5 high expressing cells are primary target of HHT, and HSPA5 may serve as a key\nmediator of the senotherapeutic activity of HHT.”\nWe also included the following sentence in the Discussion section of the revised\nmanuscript\n“Our findings suggest that the senotherapeutic effect of HHT is highly dependent on HSPA5, as its\nknockdown markedly attenuated the pro-apoptotic activity of HHT. This indicates that HHT\nrequires HSPA5 as a functional target or signaling mediator to induce cell death, and that the\nphysical interaction between HHT and HSPA5 may be critical for initiating apoptosis. In contrast,\nsimple depletion of HSPA5 expression by siRNA may allow compensatory chaperone networks or\n27\nstress-adaptive responses to preserve cell survival. Moreover, siRNA reduces protein levels\ngradually and incompletely, whereas HHT may acutely disrupt specific HSPA5 functions, including\nthose at the endoplasmic reticulum or cell surface, that are essential for maintaining proteostasis\nand stress signaling. Thus, the loss of HHT-induced cytotoxicity under HSPA5 knockdown\nconditions supports the conclusion that HSPA5 is not only associated with but also required as a\nfunctional binding partner for HHT-mediated apoptosis, highlighting a mechanistic dependency\nrather than a redundant pathway.”\n7. In Fig. 8G, the SA-β-Gal signal appears to localize to structures like bronchi or blood\nvessels, with nearly all cells within the circular region showing positive staining. Is the\nauthor attempting to suggest that all cells in this region are senescent? Furthermore,\ndoes the spatial distribution of p53 signal in this same area within the tissue?\nOur responses:\nWe appreciate the reviewer’s observation and the opportunity to clarify this point. We do\nnot intend to imply that all cells within the SA-β-Gal–positive region are senescent. SA-\nβ-Gal staining has known limitations, including background activity in metabolically\nactive or lysosome-rich cells (PMID: 17634571, 26105537, 20010931). As\nrecommended in the literature (PMID: 20010931, 39121846), senescent cell\nidentification is more reliable when supported by multiple, complementary markers. For\nthis reason, we employed additional assays, including p53 immunostaining, Western\nblotting for p53, p21, and p16, and trichrome staining. SA-β-Gal staining was performed\non frozen tissue sections, whereas p53 immunostaining was conducted on paraffin-\nembedded sections from the same cohort of mice. Because these assays were\nperformed on non-identical sections, direct spatial correlation between SA-β-Gal and\np53 signals is not possible. Nonetheless, both markers indicate the presence of\nsenescent cells in the tissue, with expected differences in their distribution due to both\nmethodological factors (different sample preparation) and biological variability between\nmarkers.\nA point-by-point response to the reviewers' comments\nReviewer #1 (Remarks to the Author):\nThe authors have adequately and thoroughly addressed the comments raised, through addition of\nnew data and edits. The data supporting HHT as a senotherapeutic and affecting WAT are strong.\nResponse: We thank Reviewer #1 for the positive comment. We are pleased that our revisions and\nnewly added data have adequately addressed the concerns raised, and we believe the reviewer’s\ncomments have substantially strengthened the manuscript.\nReviewer #2 (Remarks to the Author):\nNone\nResponse: We thank Reviewer #2 for their time and consideration of our revised manuscript.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank Reviewer #5 for their time and contribution to the peer-review process.\nWe also sincerely appreciate the Nature Communications editorial team for arranging and\nfacilitating the peer-review process, as well as for their thorough editorial handling of our\nmanuscript throughout the submission and revision process."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately and thoroughly addressed the comments raised, through addition of new data and edits. The data supporting HHT as a senotherapeutic and affecting WAT are strong.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter