# Bis(monoacylglycero)phosphate protects murine RAW 264.7 macrophages against 7-Ketocholesterol-induced apoptosis and impaired autophagy

Remi Lambert, Sherine Montillet, Clara Hennot, Agathe Diaz-Gonzalez, Marion Guichard, Emeline Cros-Perrial, Lars Petter Jordheim, Karen Gaget, Federica Calevro, Celine Luquain-Costaz, et al.

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## To cite this version:

Remi Lambert, Sherine Montillet, Clara Hennot, Agathe Diaz-Gonzalez, Marion Guichard, et al. Bis(monoacylglycero)phosphate protects murine RAW 264.7 macrophages against 7-Ketocholesterol-induced apoptosis and impaired autophagy. Journal of Steroid Biochemistry and Molecular Biology, 2026, 259, pp.106955. 10.1016/j.jsbmb.2026.106955. hal-05525347

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Submitted on 24 Feb 2026

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# Bis(monoacylglycerol)phosphate protects murine RAW 264.7 macrophages against 7-Ketocholesterol-induced apoptosis and impaired autophagy

Remi Lambert<sup>a,b</sup>, Sherine Montillet<sup>c</sup>, Clara Hennot<sup>a</sup>, Agathe Diaz-Gonzalez<sup>a</sup>, Marion Guichard<sup>a</sup>, Emeline Cros-Perrial<sup>d</sup>, Lars Petter Jordheim<sup>d</sup>, Karen Gaget<sup>e</sup>, Federica Calevro<sup>c</sup>, Celine Luquain-Costaz<sup>a,b</sup>, Isabelle Delton<sup>a,b,*</sup>

## ARTICLE INFO

**Keywords:**

Bis(monoacylglycerol)phosphate
7-Ketocholesterol
Macrophages
Apoptosis
Autophagy
Atherosclerosis

## ABSTRACT

Atherosclerosis, a leading cause of cardiovascular disease, is driven by the accumulation of oxidized low-density lipoproteins (oxLDL) in arterial walls. 7-Ketocholesterol (7KC), a major oxysterol found in oxLDL and atherosclerotic plaques, triggers multiple cell injuries including loss of lysosomal integrity, oxidative stress, apoptosis, and impaired autophagy in vascular cells. Bis(monoacylglycerol)phosphate (BMP), also known as lysobisphosphatidic acid, is a unique phospholipid concentrated in the endolysosomal compartment, known to regulate vesicle dynamics, lysosomal enzyme activities, intracellular cholesterol trafficking and its oxidative metabolism. Using a validated model of BMP enrichment in murine RAW 264.7 macrophages, we investigated whether BMP could exert protective activity against 7KC-induced damage. Our findings revealed that BMP enrichment provides comprehensive protection against 7KC at the cellular level by preserving cell viability, morphology, and neutral lipid balance. Mechanistically, BMP enrichment prevented apoptosis by maintaining mitochondrial integrity and blocking caspase activation. This was demonstrated by normalized BAX/BCL2 ratios, preserved pro-Caspase-3 levels, and reduced PARP cleavage. Remarkably, BMP enrichment also restored autophagic flux, thereby preventing the pathological accumulation of LC3-II and p62 that characterizes autophagy dysfunction. Enhanced colocalization between LC3 and BMP suggests direct functional interactions in the stress response. Gene expression analysis confirmed that BMP enrichment normalized the transcriptional dysregulation of key autophagy regulators, including *Sqstm1*, *Becn1*, and *Pink1*. Taken together, these results suggest that BMP is an endogenous protective factor that counteracts 7KC-induced cellular damage at multiple steps by regulating cell death and autophagy pathways in a coordinated manner.

## 1. Introduction

Atherosclerosis is a chronic inflammatory disease characterized by the progressive accumulation of lipids, immune cells, and fibrous elements within arterial walls, leading to plaque formation and subsequent vessel occlusion [1,2]. It represents a major cardiovascular complication

of diseases associated with elevated oxidative stress and disorders of lipid metabolism, such as type 2 diabetes, obesity, and metabolic syndrome. Among the various biochemical factors contributing to atherosclerotic lesion formation, oxidized low-density lipoproteins (oxLDL) play a central role in disease initiation and progression [3–5]. Within oxLDL particles, oxysterols represent a large family of cholesterol

Abbreviations: 7KC, 7-Ketocholesterol; ATG12/Atg12, Autophagy Related 12; ATG5/Atg5, Autophagy Related 5; BAX, BCL2 Associated X Protein; BCL2, B-cell Lymphoma 2; *Bnip3*, BCL2 Interacting Protein 3; BMP, Bismonoacylglycerophosphate; Caspase-3, Cysteine-aspartic protease 3; CTCF, corrected total cell fluorescence; *Hprt*, Hypoxanthine Phosphoribosyltransferase; LAMP1, Lysosomal-associated membrane protein 1; LBPA, Lysobisphosphatidic acid; LC3/*Map1lc3b*, Microtubule Associated Protein 1 Light Chain 3 Beta; LSD, Lysosomal storage diseases; MOMP, mitochondrial outer membrane permeabilization; OxLDL, Oxidized low-density lipoproteins; P62/*Sqstm1*, Sequestosome 1; PARP, Poly ADP-ribose polymerase; *Pink1*, PTEN-Induced Kinase 1; *Tbp*, TATA-Box Binding Protein.

* Corresponding author at: Univ Lyon, Université Claude Bernard Lyon 1, CNRS, LAGEPP UMR 5007, Villeurbanne F-69622, France.

E-mail address: isabelle.delton@insa-lyon.fr (I. Delton).

<footnote><sup>a</sup> Univ Lyon, Université Claude Bernard Lyon 1, CNRS, LAGEPP UMR 5007, Villeurbanne F-69622, France</footnote>

<footnote><sup>b</sup> INSA Lyon, Villeurbanne F-69621, France</footnote>

<footnote><sup>c</sup> SATT, Ouest Valorisation, Rennes F-35700, France</footnote>

<footnote><sup>d</sup> Univ Lyon, Université Claude Bernard Lyon 1, INSERM U1052, CNRS 5286, Centre Léon Bérard, Centre de Recherche en Cancérologie de Lyon, Lyon F-69373, France</footnote>

<footnote><sup>e</sup> INRAE, INSA Lyon, BF2I UMR0203, Villeurbanne F-69621, France</footnote>
oxidation products that regulate various biological activities, including cholesterol homeostasis, cellular oxidative stress, and inflammation in vascular cells [6–8]. 7-Ketocholesterol (7KC), an abundant and biologically active oxysterol derived from non-enzymatic oxidation of cholesterol, is found in several common foods [9], as well as in oxLDL and atherosclerotic plaques. It has been shown to promote the formation of macrophage foam cells, perturbation of endothelial integrity, mono-cyte adhesion and transmigration, and plaque progression and instability [7,10,11]. 7KC has been demonstrated to exhibit potent cytotoxic and pro-atherogenic properties through multiple cellular mechanisms, including mitochondrial dysfunction, endoplasmic reticulum stress, oxidative stress generation, and lysosomal damage [12–14]. Within vascular cells, including endothelial cells, smooth muscle cells, and macrophages, 7KC exposure triggers complex cellular responses involving both autophagy and apoptosis [15–17]. The term "oxia-poptophagy" has been proposed to describe a specific form of cell death characterized by the simultaneous activation of oxidative stress, apoptotic cascades, and autophagic processes induced by oxysterols such as 7KC [18,19]. Autophagy exhibits paradoxical effects in atherosclerosis. Under mild oxidative stress, it protects against apoptosis by removing damaged mitochondria and maintaining plaque stability [17, 20], whereas severe oxidative stress impairs autophagic flux through lysosomal damage, leading to cell death that favors plaque progression [21,22]. In macrophages, autophagy also regulates foam cell formation and cholesterol efflux [23–25].

The lysosomal compartment plays a pivotal role in cellular lipid metabolism and the response to 7KC-induced stress. The integrity and functional capacity of lysosomes are particularly critical in cells exposed to high lipid loads, such as macrophage-derived foam cells in atherosclerotic plaques [26]. 7KC has been documented to induce lysosomal dysfunction, characterized by lysosomal membrane permeabilization [27,28], cathepsin B and D inhibition [28], and impaired autophagic flux [17,22,29]. The endolysosomal compartment is characterized by its enrichment in bis(monoacylglycerol)phosphate (BMP), also known as lysobisphosphatidic acid (LBPA). BMP is a phospholipid with distinctive features, including an unusual *sn*-1-glycerophosphate-*sn*-1'-glycerol (*sn*-1:*sn*-1') backbone stereochemistry and abundance in esterified long-chain unsaturated fatty acids, particularly oleic acid and docosahexaenoic acid [30,31]. BMP, representing up to 70 % of total phospholipids within the intraluminal membranes of late endosomes and lysosomes [32,33], plays essential regulatory roles in multiple aspects of lysosomal function. BMP serves as a cofactor for several lysosomal hydrolases, including sphingomyelinase and glucocerebrosidase, enhancing their catalytic activity and enabling efficient substrate processing within the acidic lysosomal environment [34–37]. Moreover, BMP influences the spatial organization of lysosomal membranes and the formation of intraluminal vesicles, which are critical for the sequestration and degradation of membrane proteins and lipids [31,33, 36,38]. BMP has also been widely described for its role in regulating cholesterol export from the endolysosomal compartment [30,39,40]. In previous studies, we demonstrated that BMP regulates cholesterol metabolism and trafficking in murine RAW macrophages exposed to native or oxLDL [41,42]. We also showed that BMP exerts protective effects against oxLDL-induced cellular stress by reducing oxysterol formation and inhibiting apoptosis in macrophages [43,44]. More recently, a role of BMP in the regulation of the autophagy-apoptosis crosstalk has also been reported [36,45].

Given the growing evidence for the involvement of endolysosomal BMP in cellular functions related to cholesterol homeostasis, apoptosis, and autophagy, we investigated the potential protective effects of BMP against 7KC-induced cellular damage. Using a previously validated model of BMP enrichment in RAW macrophage [42,46], we examined the effects of BMP on 7KC-induced cytotoxicity, characterized the associated cell death mechanisms (i.e. apoptosis and autophagy), and explored the underlying molecular pathways.

# 2. Materials and methods

## 2.1. Materials

### 2.1.1. Cell culture materials

Murine RAW 264.7 macrophages were obtained from the American Type Culture Collection. Minimum Essential Medium (MEM) and non-essential amino acids (NEAA) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) and penicillin-streptomycin (PS) were obtained from Dutscher, and mycoplasma elimination reagent nanomycopulitine from Biowest. 1,2-Dioleoyl-*sn*-glycero-3-phospho-(1'-rac-glycerol) sodium salt (DOPG) and 7KC were purchased from Avanti Polar Lipids.

### 2.1.2. Antibodies

The following primary antibodies were used: rabbit polyclonal anti-Actin (Sigma-Aldrich, #A2066), rabbit monoclonal anti-ATG5 (Cell Signaling Technology, #12994), rabbit polyclonal anti-BAX (Cell Signaling Technology, #2772), rabbit monoclonal anti-BCL2 (Abcam, #AB182858), rabbit monoclonal anti-BECLIN1 (Cell Signaling Technology, #3495), rabbit monoclonal anti-Caspase-3 (Cell Signaling Technology, #14220), rabbit polyclonal anti-LAMP1 (Sigma-Aldrich, #L1418), mouse monoclonal anti-LBPA (Sigma-Aldrich, #MABT837), rabbit polyclonal anti-LC3 (Sigma-Aldrich, #L7543), rabbit monoclonal anti-p62 (Abcam, #AB109012), and rabbit polyclonal anti-PARP (Cell Signaling Technology, #9542). Secondary antibodies included HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology, #7074), Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, #A11034), and Alexa Fluor 546-conjugated donkey anti-mouse IgG (Life Technologies, #A10036).

## 2.2. Culture and treatments of murine RAW 264.7 macrophages

Murine RAW 264.7 macrophages were routinely cultured as monolayers in MEM supplemented with 10 % (v/v) FBS, 2 % (v/v) PS, 1 % (v/v) NEAA, and 1 % (v/v) nanomycopulitine, at 37 °C in a humidified atmosphere containing 5 % CO₂, and sub-cultured twice a week. For experiments, cells were seeded at a density of 4 × 10⁴ viable cells/cm² in complete MEM and allowed to adhere overnight. BMP enrichment of RAW macrophages was performed by supplementation with its precursor DOPG [30,46]. DOPG liposomes were prepared extemporaneously from a 10 mg/mL stock solution in chloroform using the thin-film hydration method, as previously described [42,46]. Cells were maintained in control MEM (referred to as non-enriched cells) or in MEM supplemented with 30 µM DOPG liposomes for 24 h (referred to as BMP-enriched cells). At this concentration, DOPG internalized by RAW macrophages is completely converted into BMP, which concentrates in the endolysosomal compartment [42,46]. Non-enriched and BMP-enriched cells were then exposed to various concentrations of 7KC in ethanolic solution (12.5, 25, or 50 µM; with ethanol not exceeding 0.5 % (v/v) final) for 24 h. Control conditions correspond to non-enriched, unexposed cells.

## 2.3. Cell morphology and viability

Cell morphology was assessed using an Eclipse TS100 inverted phase-contrast microscope (Nikon) equipped with a DS-Fi2 high-definition color camera and a DS-L3 standalone control unit. Cell viability was quantified by colorimetric MTS assay using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega), according to the manufacturer's instructions. Absorbance was measured at 490 nm using a Spark® multimode microplate reader (Tecan). Cell viability was expressed as a percentage relative to control, and data were collected from at least three independent experiments.
## 2.4. Fluorescence staining, imaging and analysis

Cells cultured on coverslips were fixed with 4% (w/v) paraformaldehyde (PFA; Dutscher) in PBS for 20 min at RT, and washed three times with PBS. To visualize lipid droplets, cells were stained with 0.5 µM Nile Red (Sigma-Aldrich) in PBS for 30 min at room temperature (RT) in the dark, rinsed three times with PBS, counterstained with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen) for 20 min at RT in the dark, and washed again with PBS. For cholesterol visualization, cells were stained with 100 µg/mL Filipin III (Sigma-Aldrich) in PBS for 3 h at RT in the dark, rinsed three times with PBS, counterstained with 1:10,000 (v/v) SYBR™ Green I Nucleic Acid Gel Stain (ThermoFisher Scientific) in PBS for 5 min at RT in the dark, and washed again with PBS. For immunofluorescence staining of BMP, LC3, and p62, cells were permeabilized with 0.1% (v/v) Triton X-100 (Sigma-Aldrich) in PBS for 12 min at RT, washed three times with PBS, and blocked with 0.1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) in PBS for 30 min at RT. Primary antibodies against BMP (1:100), LC3 (1:200), or p62 (1:400) were applied for 1 h at RT, followed by three PBS washes. Cells were then incubated with Alexa Fluor 488- or Alexa Fluor 546-conjugated secondary antibodies (1:1000) for 1 h at RT in the dark. All antibody dilutions were prepared in blocking solution. Cells were rinsed three times with PBS, counterstained with DAPI for 20 min at RT in the dark, and washed again with PBS. Coverslips were mounted on glass slides with Fluoromount mounting medium (Sigma-Aldrich) and imaged.

Images were acquired using a Leica DM6B fluorescence microscope (Leica Microsystems) with Z-stack acquisition and real-time deconvolution performed with Thunder™ technology to reduce blur and light scattering. Image analysis was carried out using LAS X (Leica Microsystems) and ImageJ/Fiji software. Fluorescence intensity was quantified by calculating the corrected total cell fluorescence (CTCF) after background subtraction. Colocalization analysis was performed using the Colocalization Test plug-in [47], and Pearson's correlation coefficient was used as the colocalization index. Data were collected from three independent experiments.

## 2.5. Quantification of apoptotic and necrotic cells by flow cytometry

Apoptosis was assessed using the BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer's instructions. Annexin V-FITC and propidium iodide (PI) fluorescence were measured using a BD Accuri™ C6 Plus flow cytometer (BD Biosciences). At least 50,000 events were acquired per sample and analyzed using BD CSampler Plus software (BD Biosciences). Cell populations were distinguished as follows: live cells (Annexin V −/PI −), early apoptotic cells (Annexin V +/PI −), late apoptotic cells (Annexin V +/PI +), and necrotic cells (Annexin V −/PI +). Total apoptosis was calculated as the sum of early and late apoptotic populations. Data were collected from four independent experiments.

## 2.6. Quantification of proteins by polyacrylamide gel electrophoresis and western blot analyses

Cells were lysed in RIPA buffer (Merck) supplemented with protease and phosphatase inhibitor cocktail (Roche) for 30 min on ice, with gentle vortexing every 5 min to ensure complete lysis. Lysates were clarified by centrifugation at 16,000 × g for 20 min at 4 °C. For each sample, 30 µg of total protein were denatured in Laemml buffer (Bio-Rad) containing 10% (v/v) 2-mercaptoethanol (Merck) at 95 °C for 5 min and separated by SDS-PAGE on 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad). Proteins were then transferred onto PVDF membranes (ThermoFisher Scientific). Membranes were blocked with either 5% (w/v) nonfat milk or 5% (w/v) BSA (Sigma-Aldrich) in Tris-buffered saline containing 0.05% Tween-20 (TBST), depending on the antibody. Membranes were then incubated overnight at 4 °C with primary antibodies diluted in either 5% (w/v) nonfat milk or 5% (w/v)

BSA in TBST, as follows: anti-Actin (1:1000, milk), anti-ATG5 (1:1000, BSA), anti-BAX (1:1000, BSA), anti-BCL2 (1:1500, milk), anti-BECLIN1 (1:1000, BSA), anti-Caspase-3 (1:1000, BSA), anti-LAMP1 (1:300, milk), anti-LC3 (1:1000, milk), anti-p62 (1:3000, milk), and anti-PARP (1:1500, milk). After three washes in TBST, membranes were incubated with HRP-conjugated secondary antibody (1:3000) for 1 h at RT. After three additional washes in TBST, signals were detected using the Clarity Max chemiluminescence kit (Bio-Rad) and imaged with a ChemiDoc XRS+ system (Bio-Rad). Band intensities were quantified using ImageJ/Fiji software, and protein expression levels were normalized to Actin for comparison across conditions. Protein expression was expressed as a percentage relative to untreated control cells, and data were collected from at least three independent experiments.

## 2.7. Quantification of gene expression by qRT-PCR

Total RNA was extracted using TRIzol reagent (Ambion) according to the manufacturer's protocol. RNA samples were subsequently treated with RQ1 RNase-Free DNase I (Promega) to eliminate genomic DNA contamination. First-strand cDNA synthesis was performed using the SuperScript™ III First-Strand Synthesis System (Life Technologies) with random hexamers and oligo(dT)₂₀ primers (Life Technologies). Real-time PCR was carried out on a CFX96 Real-Time PCR Detection System (Bio-Rad) using 1:5 diluted cDNA and the LightCycler® 480 SYBR Green I Master Mix (Roche), following the manufacturer's instructions. The primer sequences are listed in Table 1. mRNA expression levels were quantified relative to the *Tbp* gene, identified as the most stable reference gene by the BestKeeper software [48] when compared with the *Hprt* gene. Relative gene expression levels were determined using the Pfaffl method [49], which accounts for amplification efficiency and crossing point differences between test and control samples. For each condition, gene expression was normalized to *Tbp* and expressed as fold induction relative to untreated control cells. All qRT-PCR reactions were performed in technical triplicates, and data were collected from four independent experiments.

## 2.8. Statistical analysis

All experimental data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using Jamovi (version 2.6.24.0). Fluorescence (intensity and colocalization) and qRT-PCR data were analyzed using linear mixed-effects models (GAMLj module) with treatments as fixed effects and experimental/biological replicates as random intercepts, followed by Bonferroni correction for multiple comparisons. MTS, flow cytometry, and western blot data were analyzed

<table><caption>**Table 1**

Oligonucleotide primers used for qRT-PCR. Forward (F) and reverse (R) primer sequences and amplicon lengths are provided for each gene.</caption><thead><tr><th>Gene</th><th>Primer</th><th>Amplicon length (bp)</th></tr></thead><tbody><tr><td>Atg5</td><td>F: 5'-AGTCGCCCCTGAAGATGGAG-3'<br>R: 5'-GCAGAGGGGTTTCCAGCATT-3'</td><td>77</td></tr><tr><td>Atg12</td><td>F: 5'-AAATGGGCTGTGGAGCGAAC-3'<br>R: 5'-CCATCACTGCCAAAACACTCATA-3'</td><td>167</td></tr><tr><td>Becn1</td><td>F: 5'-AAACCAGGAGAGACCCAGGAG-3'<br>R: 5'-TTTCTGTAGACATCATCCTGGCTGG-3'</td><td>118</td></tr><tr><td>Bnip3</td><td>F: 5'-TTCTCACTGTGACAGCCAC-3'<br>R: 5'-TCTTCCTCAGACAGAGTGCT-3'</td><td>108</td></tr><tr><td>Hprt</td><td>F: 5'-TTGCTGACCTGCTGGATTAC-3'<br>R: 5'-AGTTGAGAGATCATCTCCAC-3'</td><td>148</td></tr><tr><td>Map1lc3b</td><td>F: 5'-AAGATCCCAGTGATTATAGAGCGA-3'<br>R: 5'-TGCAAGCGCCGTCTGATTAT-3'</td><td>128</td></tr><tr><td>Sqstm1</td><td>F: 5'-CCTCAGCCCTCTAGGCATTG-3'<br>R: 5'-TTCTGGGGTAGTGGGTGTCA-3'</td><td>82</td></tr><tr><td>Pink1</td><td>F: 5'-AGTGGGACTCAGATGGCTGT-3'<br>R: 5'-CCATTGCCACCACGCTCTAC-3'</td><td>121</td></tr><tr><td>Tbp</td><td>F: 5'-TTCACATCACAGCTCCCCAC-3'<br>R: 5'-TGGTGTGCACAGGAGCCAAG-3'</td><td>138</td></tr></tbody></table>
using one-way ANOVA followed by Tukey's HSD post hoc test. Assumptions of normality and homogeneity of variance were verified using Shapiro-Wilk tests and residual plot inspection. Statistical significance was set at *p* < 0.05.

# 3. Results

## 3.1. Assessment of the cytoprotective effect of BMP enrichment on 7KC-induced cellular toxicity

Cell growth and morphology were first evaluated through phase-contrast microscopic examination (Fig. 1A). Cells exposed to 50 µM 7KC alone for 24 h displayed substantial morphological alterations characteristic of cell death, including reduced cell number, pronounced cell shrinkage, fragmentation, and loss of adherence compared to unexposed ones. BMP enrichment alone had no effect but substantially preserved normal cell growth and morphology upon 7KC exposure. Quantification of viable cells using the MTS colorimetric assay revealed that, in non-enriched cells, 7KC induced a dose-dependent reduction in cell viability following 24 h exposure (Fig. 1B, left). At 12.5 µM, 7KC exhibited minimal cytotoxicity (~90 % viability), whereas exposure to 50 µM resulted in substantial cell death (~20 % viability). An intermediate effect was observed at 25 µM (~65 % viability). Based on these findings, concentrations of 12.5, 25, and 50 µM were selected to represent minimally, moderately, and highly cytotoxic conditions, respectively, for subsequent experiments. Notably, in BMP-enriched cells, viability was fully restored following exposure to 25 µM 7KC and partially restored (~60 % viability) following exposure to 50 µM 7KC (Fig. 1B, right).

These findings demonstrate that BMP enrichment effectively attenuates 7KC-induced cytotoxicity in murine RAW 264.7 macrophages, indicating significant cytoprotective potential against oxysterol-mediated cellular injury.

## 3.2. BMP enrichment prevents 7KC-induced disruption of lipid homeostasis

Given the established role of oxysterols in disrupting cellular lipid homeostasis, we investigated whether BMP enrichment could modulate 7KC-induced lipid dysregulation. Nile Red staining revealed distinct

alterations in lipid droplet distribution and content across experimental conditions (Fig. 2A). Control cells displayed well-defined Nile Red staining, predominantly localized in discrete cytoplasmic lipid droplets. In contrast, 7KC exposure markedly reduced Nile Red fluorescence intensity and disrupted the characteristic lipid droplet pattern in non-enriched cells, resulting in faint and irregularly distributed staining. BMP enrichment restored a Nile Red staining profile closely resembling that of control cells, as evidenced by the recovery of both fluorescence intensity and organized lipid droplet morphology. Quantitative analysis confirmed these observations (Fig. 2B), as indicated by a significant 3-fold decrease in Nile Red fluorescence intensity (72,700 ± 6200 a.u. vs. 198,800 ± 9900 a.u.) in non-enriched cells exposed to 7KC compared to control cells. BMP enrichment attenuated this lipid depletion, maintaining Nile Red fluorescence intensity at levels substantially higher (121,600 ± 11,200 a.u.) than those observed in non-enriched cells exposed to 7KC. Notably, BMP enrichment alone displayed no changes in Nile Red fluorescence intensity or cellular distribution compared to control conditions (data not shown). Filipin staining revealed no significant differences in free cholesterol distribution or fluorescence intensity across experimental conditions (data not shown).

These findings demonstrate that BMP enrichment effectively protects against 7KC-induced neutral lipid depletion in macrophages, thereby maintaining lipid homeostasis under oxysterol-induced stress.

## 3.3. BMP enrichment attenuates 7KC-induced apoptotic signaling

To assess the impact of BMP enrichment on 7KC-induced apoptosis, we first performed flow cytometry analysis using Annexin V-FITC/PI double staining to distinguish live, apoptotic, and necrotic cell populations (Fig. 3A-B). In non-enriched cells, 7KC induced a concentration-dependent shift from viable toward apoptotic and necrotic cells. At 25 µM 7KC, apoptosis increased markedly, accompanied by a visible but not statistically significant increase in necrosis. At the highest concentration, 7KC triggered substantial cell death, with a strong increase in both apoptotic and necrotic cells. Remarkably, BMP enrichment substantially attenuated 7KC-induced cell death by both apoptosis and necrosis across all concentrations tested. At 50 µM 7KC, the apoptotic and necrotic populations in BMP-enriched cells were reduced by 2-fold and 5-fold, respectively, compared to non-enriched cells.

To further characterize apoptotic signaling, molecular apoptotic

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** BMP enrichment protects RAW 264.7 macrophages against 7KC-induced cytotoxicity. (A) Representative phase-contrast images of RAW 264.7 macrophages exposed to 50 µM 7KC for 24 h without or with BMP enrichment. Control condition corresponds to non-enriched, unexposed cells. Magnification, × 200. (B) Cell viability assessed by MTS assay after exposure to various concentrations of 7KC (12.5–50 µM) for 24 h without or with BMP enrichment. Data are expressed as percentage of control and represent mean ± SEM from at least three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's HSD post hoc test. \*p < 0.05 vs. control; \#p < 0.05 vs. corresponding non-enriched condition.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** BMP enrichment prevents 7KC-induced neutral lipid depletion in RAW 264.7 macrophages. Cells were exposed to 25 µM 7KC for 24 h without or with BMP enrichment. (A) Representative fluorescence microscopy images of Nile Red-stained lipid droplets (red) and DAPI-stained nuclei (blue). Control condition corresponds to non-enriched, unexposed cells. Scale bars, 10 µm. (B) Quantification of Nile Red fluorescence intensity expressed as CTCF in arbitrary units (a.u.). Each data point represents the mean CTCF per image (14 images per condition per experiment). Data were acquired from 42 images across three independent experiments (>1700 cells per condition), with colors indicating independent replicates. Box plots show median, interquartile range, and full distribution. Statistical analysis was performed using linear mixed-effects models followed by Bonferroni correction. \*p < 0.05 vs. control; \#p < 0.05 vs. corresponding non-enriched condition.</figcaption></figure>

markers were examined through western blot analyses of PARP cleavage, BCL2 family protein expression, and pro-Caspase-3 levels (Fig. 3C). In non-enriched cells, PARP cleavage, an established hallmark of apoptotic execution, remained relatively stable at 12.5 and 25 µM 7KC but exhibited a sharp increase at 50 µM 7KC. Quantification of the cleaved PARP/total PARP ratio revealed an approximately 5-fold increase at 50 µM 7KC compared to controls (Fig. 3D). Notably, BMP enrichment prevented 7KC-induced PARP cleavage, restoring the cleaved PARP/total PARP ratio to the level of unexposed cells. Analysis of pro-Caspase-3, the inactive precursor of the principal executioner caspase, revealed a concentration-dependent reduction in non-enriched cells following 7KC exposure, consistent with proteolytic activation and apoptotic execution (Fig. 3E). BMP enrichment substantially preserved pro-Caspase-3 levels at values comparable to controls across all 7KC concentrations. Examination of BCL2 family proteins, key regulators of mitochondrial outer membrane permeabilization (MOMP) and apoptotic commitment, revealed complementary findings (Fig. 3F). In non-enriched cells, the anti-apoptotic protein BCL2 progressively decreased in response to rising 7KC concentrations, while the pro-apoptotic protein BAX remained elevated above control levels at all doses. Consequently, the BAX/BCL2 ratio, a critical determinant of apoptotic susceptibility, increased substantially in non-enriched cells under 7KC treatment. In contrast, BMP enrichment maintained the BAX/BCL2 ratio at levels comparable to controls across all 7KC concentrations.

These results demonstrate that 7KC triggers strong apoptotic signaling through multiple converging pathways, including PARP cleavage, Caspase-3 activation, and disruption of the BAX/BCL2 ratio. BMP enrichment provides substantial protection against 7KC-induced apoptosis at each of these regulatory checkpoints, effectively preventing both mitochondrial and executioner phases of the apoptotic cascade.

### 3.4. BMP enrichment prevents 7KC-induced autophagy and autophagic flux impairment

We next examined the expression and cellular distribution of key autophagy markers: LC3 (a marker and mediator of autophagosome formation) and p62 (an autophagy adaptor linking ubiquitinated cargo to LC3).

Immunofluorescence imaging revealed that exposure to 25 µM 7KC markedly altered the cellular distribution of LC3, displaying a disorganized fluorescence pattern compared to control cells (Fig. 4A). Quantitative analysis demonstrated a significant increase in LC3 fluorescence intensity in 7KC-exposed cells relative to controls (110,800 ± 6400 a.u. vs. 53,000 ± 2400 a.u.) (Fig. 4B). Importantly, BMP enrichment effectively prevented this LC3 accumulation, reducing fluorescence intensity to near-control levels (58,100 ± 3300 a.u.) and restoring a normal LC3 distribution pattern. Colocalization analysis using Pearson's correlation coefficient revealed increased LC3/BMP association (0.713 ± 0.012 vs. 0.648 ± 0.014) in 7KC-exposed cells compared to controls, suggesting a direct involvement of BMP in autophagy regulation (Fig. 4C). As for p62, a modified distribution pattern and a significant increase in fluorescence intensity were observed in cells exposed to 7KC compared to controls (20,700 ± 2900 a.u. vs. 10,600 ± 1500 a.u.) (Fig. 4D-E). The concomitant elevation of both LC3 and p62 is consistent with autophagosome accumulation due to impaired autophagosome-lysosome fusion following 7KC exposure [50]. Notably, in BMP-enriched cells, p62 levels were fully normalized (7600 ± 700 a.u.), indicating restoration of autophagic flux. Of note, BMP enrichment alone displayed no changes in LC3 or p62 fluorescence intensity, cellular distribution, or LC3/BMP colocalization compared to control conditions (data not shown).

Western blot analyses were performed to measure LC3 conversion and p62 expression (Fig. 4F). The cytosolic form LC3-I showed a slight dose-dependent accumulation in non-enriched cells exposed to 7KC, while the membrane-bound form LC3-II exhibited a pronounced dose-dependent accumulation. Consequently, the LC3-II/LC3-I ratio, a critical indicator of autophagic flux, rose progressively, reaching nearly a 4-
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** BMP enrichment attenuates 7KC-induced apoptosis in RAW 264.7 macrophages. Cells were exposed to various concentrations of 7KC (12.5–50 µM) for 24 h, without or with BMP enrichment. (A) Representative flow cytometry dot plots of Annexin V-FITC/PI double-stained cells. Control condition corresponds to non-enriched, unexposed cells. (B) Quantification of cell populations (live, apoptotic, and necrotic cells) expressed as percentage of total cells. (C) Representative western blot analysis of apoptosis-related proteins (total PARP, cleaved PARP, pro-Caspase-3, BCL2, and BAX). (D) Quantification of cleaved PARP/total PARP ratio expressed as fold change relative to control. (E) Quantification of pro-Caspase-3 protein expression normalized to Actin, expressed as percentage of control. (F) Quantification of BAX/BCL2 ratio expressed as fold change relative to control. For panels B, D, E, and F: Data represent mean ± SEM from at least three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's HSD post hoc test. \*p < 0.05 vs. control; \#p < 0.05 vs. corresponding non-enriched condition.</figcaption></figure>

fold increase at the highest 7KC concentration (Fig. 4G). Remarkably, BMP enrichment significantly attenuated this response across all 7KC concentrations tested, reducing the LC3-II/LC3-I ratio by 30 % at 12.5 and 25 µM 7KC and by 47 % at 50 µM. Similarly, p62 expression

increased in a dose-dependent manner in non-enriched cells following 7KC exposure, reaching up to a 6-fold elevation at 50 µM compared to unexposed cells (Fig. 4H), whereas the increase was limited to 2-fold in BMP-enriched cells. The expression of additional autophagy markers
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** BMP enrichment prevents 7KC-induced autophagy dysregulation and restores autophagic flux in RAW 264.7 macrophages. Cells were exposed to 25 µM 7KC for 24 h without or with BMP enrichment. Control condition corresponds to non-enriched, unexposed cells. (A) Representative fluorescence microscopy images of LC3 (green), BMP (red), and DAPI-stained nuclei (blue). Scale bars, 10 µm. (B) Quantification of LC3 fluorescence intensity expressed as CTCF in arbitrary units (a.u.). (C) Colocalization analysis between LC3 and BMP expressed as Pearson's correlation coefficient. (D) Representative fluorescence microscopy images of p62 (red) and DAPI-stained nuclei (blue). Scale bars, 5 µm. (E) Quantification of p62 fluorescence intensity expressed as CTCF in arbitrary units (a.u.). For panels B, C, and E: Each data point represents the mean CTCF per image (7 images per condition per experiment). Data were acquired from 21 images across three independent experiments (>700 cells per condition), with colors indicating independent replicates. Box plots show median, interquartile range, and full distribution. Statistical analysis was performed using linear mixed-effects models followed by Bonferroni correction. (F) Representative western blot analysis of autophagy-related proteins (LAMP1, BECLIN1, ATG5-ATG12 heterodimer, p62, and LC3-I/II) after exposure to 7KC (12.5–50 µM) for 24 h without or with BMP enrichment. (G) Quantification of LC3-II/LC3-I ratio expressed as fold change relative to control. (H) Quantification of p62 protein expression normalized to Actin, expressed as percentage of control. For panels G and H: Data represent mean ± SEM from at least three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's HSD post hoc test. \*p < 0.05 vs. control; \#p < 0.05 vs. corresponding non-enriched condition.</figcaption></figure>
including BECLIN1 (involved in autophagy initiation), the ATG5-ATG12 heterodimer (involved in autophagosome membrane elongation), and LAMP1 (a lysosomal membrane protein) remained unchanged across all conditions (Fig. 4F), indicating that the 7KC-induced autophagy dysfunction occurs independently of alterations in autophagy initiation, autophagosome membrane expansion, or lysosomal biogenesis. Together, these findings demonstrate that BMP enrichment effectively prevents 7KC-induced autophagosome accumulation and restores autophagic flux in RAW 264.7 macrophages, highlighting a protective role for BMP in maintaining autophagy homeostasis under oxysterol-induced stress.

We next examined transcriptional regulation of key autophagy-related genes by qRT-PCR analysis. Consistent with protein-level observations, *Sqstm1* (encoding p62) mRNA expression increased in a dose-dependent manner from 1.7- to 4.0-fold in non-enriched cells exposed to 7KC (Fig. 5A). BMP enrichment fully reversed this 7KC-induced *Sqstm1* overexpression. Similarly, *Becn1* (encoding BECLIN1) expression was slightly but significantly increased 1.5-fold in non-enriched cells at

25 µM and 50 µM 7KC, an effect fully reversed by BMP enrichment (Fig. 5B). *Pink1* (encoding PINK1, a key regulator of mitophagy) was significantly upregulated 1.4- to 1.7-fold at 12.5 µM and 25 µM 7KC in non-enriched cells, suggesting activation of mitochondrial quality control pathways (Fig. 5C). At 50 µM 7KC, *Pink1* expression returned to near-baseline levels, potentially reflecting a shift in cellular response mechanisms at higher toxic concentrations. Importantly, BMP enrichment restored *Pink1* expression to control levels at 12.5 µM and 25 µM 7KC, indicating that membrane stabilization may alleviate mitochondrial dysfunction. mRNA levels of other autophagy-related genes, including *Atg5* (encoding ATG5), *Atg12* (encoding ATG12), *Map1lc3b* (encoding LC3), and *Bnip3* (encoding BNIP3, involved in mitophagy and apoptosis), remained largely unchanged across all experimental conditions (Fig. 5D-G). Together, these findings demonstrate that 7KC induces selective transcriptional reprogramming of autophagy genes, particularly those involved in autophagy initiation (*Becn1*), cargo recognition (*Sqstm1*), and mitochondrial quality control (*Pink1*), and that BMP enrichment effectively counteracts these transcriptional alterations,

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

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**Fig. 5.** BMP enrichment reverses 7KC-induced transcriptional upregulation of selective autophagy genes in RAW 264.7 macrophages. Cells were exposed to various concentrations of 7KC (12.5–50 µM) for 24 h, without or with BMP enrichment. (A–G) Relative mRNA levels of *Sqstm1*, *Becn1*, *Pink1*, *Map1lc3b*, *Atg5*, *Atg12*, and *Bnip3*. Control condition corresponds to non-enriched, unexposed cells. Data are expressed as fold change relative to control and represent mean ± SEM from four independent experiments. Statistical analysis was performed using linear mixed-effects models followed by Bonferroni correction. \*p < 0.05 vs. control; \#p < 0.05 vs. corresponding non-enriched condition.
thereby maintaining autophagic and mitochondrial homeostasis under 7KC-induced stress.

## 4. Discussion

This study demonstrates that BMP enrichment effectively counteracts 7KC-induced cellular damage in RAW macrophages. BMP enrichment indeed protects against cytotoxicity, restores lipid homeostasis, inhibits apoptotic signaling cascades, and prevents autophagy dysfunction through the regulation of key autophagy-related genes. Noteworthy, these protective effects were observed at 7KC concentrations comparable to those detected in atherosclerotic plaques [51,52]. Previous studies have shown that omega-3 and omega-9 fatty acids, as well as α-tocopherol and dietary polyphenols, reduce 7KC-induced cytotoxicity/apoptosis through mitochondrial and peroxisomal protection and attenuation of oxidative stress [53–57]. Our findings extend this body of work by demonstrating that BMP exerts protective effects against 7KC through mechanisms closely linked to endolysosomal function and autophagy regulation (Fig. 6).

In various cellular macrophage models, 7KC has been shown to induce lipid accumulation, including free cholesterol and intracellular lipid droplets [58–61]. In contrast, we observed a significant reduction in lipid droplet content in RAW macrophages exposed to 7KC, as assessed by Nile Red staining. This divergent response may reflect differences in experimental conditions such as cell type, fluorescent probe, or 7KC concentration. It may also stem from cell type-specific lipid metabolic reprogramming, given that 7KC has been shown to modulate lipid metabolism genes, thereby decreasing triacylglycerol and cholesterol biosynthesis in cardiac cells [62]. BMP enrichment restored neutral lipid content to near-control levels, which is consistent with our previous observation that BMP enrichment favors foam cell formation in RAW macrophages exposed to oxLDL [42]. Considering the critical importance of lipid homeostasis balance in macrophage function, the capacity of BMP to counteract 7KC-mediated lipid alterations may contribute to potential atheroprotective effects. The underlying mechanisms may be linked to the role of BMP in maintaining lysosomal

function, including lipid catabolism and cholesterol trafficking [31,36].

The anti-apoptotic effects of BMP enrichment at multiple regulatory checkpoints are noteworthy. Restoration of the BAX/BCL2 balance indicates that BMP preserves mitochondrial integrity and prevents MOMP, a critical point of no return in the intrinsic apoptotic pathway [63,64]. The preservation of pro-Caspase-3 levels and the reduction in PARP cleavage demonstrate that BMP inhibits caspase activation at both the initiator and effector stages. BMP may confer anti-apoptotic protection through complementary pathways. By preserving lysosomal membrane integrity, BMP may prevent cathepsin release and subsequent activation of caspase-dependent or -independent cell death pathways [16,65,66]. As a cofactor for sphingomyelinase and glucocerebrosidase [36,37], BMP may also ensure efficient processing of complex lipids that accumulate under stress, preventing the accumulation of pro-apoptotic ceramide and glycosphingolipids during 7KC exposure.

A central finding of our study is that BMP enrichment effectively prevents 7KC-induced autophagosome accumulation and restores autophagic flux. The concomitant elevation of both LC3-II and p62 following 7KC exposure is characteristic of impaired autophagosome-lysosome fusion rather than enhanced autophagy induction [50]. 7KC-mediated autophagy blockade may also involve disruption of SNARE proteins, key components of the fusion machinery, through aberrant sequestration in cholesterol-enriched endolysosomal membranes under oxysterol stress [67]. Noteworthy, BMP enrichment was reported to facilitate cholesterol clearance from the endolysosomal compartment [39], a mechanism that could help restore proper SNARE protein function. Autophagy has been shown to regulate cholesterol hydrolysis and efflux via lysosomal acid lipase in macrophages [23,25]. *In vivo* studies have highlighted autophagy dysfunction in atherosclerosis and demonstrated that autophagy exerts an important protective role by limiting inflammation, apoptosis, and plaque progression [22, 24]. Hence, by restoring autophagic flux and maintaining lysosomal integrity, BMP enrichment may enhance the activity of these pathways and eventually promote atheroprotection. The enhanced LC3-BMP colocalization observed after exposure to 7KC suggests a direct functional interaction between autophagosomal membranes and

<figure><img src="image_15.png" /><figcaption>**Fig. 6.** Graphical abstract as a summary.</figcaption></figure>
BMP-enriched endolysosomal compartments. As a major phospholipid constituent of late endosome/lysosome intraluminal membranes [32] and, given its unique structure and abundance in unsaturated fatty acids (including oleic and docosahexaenoic acids [30,31]), BMP could facilitate membrane fusion events that are essential for autophagosome cargo degradation. Our findings are consistent with emerging evidence that BMP plays central regulatory roles in autophagy, cargo sorting, cholesterol homeostasis, and inter-organelle communication [31,36,45].

The dose-dependent transcriptional upregulation of *Sqstm1* and *Becn1* following 7KC exposure, and their normalization by BMP enrichment, revealed an additional layer of regulatory complexity. The coupling of *Sqstm1* transcriptional induction with p62 accumulation suggests the activation of a pathological positive feedback loop [68,69]: p62 accumulation resulting from impaired autophagic flux promotes KEAP1 sequestration, leading to NRF2 stabilization and nuclear translocation. Since *Sqstm1* is a direct NRF2 gene target, this contributes to further upregulating p62 expression, creating a self-reinforcing cycle characteristic of severe autophagy dysfunction. BMP enrichment disrupts this loop by restoring autophagic flux, thereby preventing p62 accumulation and reducing compensatory transcriptional responses. This restoration of the autophagy-apoptosis balance represents a key mechanism by which BMP may promote cell survival under oxysterol-induced stress [21,70]. The transcriptional induction of *Pink1* at lower 7KC concentrations provides additional insights into mitochondrial stress responses. PINK1 initiates mitophagy when mitochondrial membrane potential is compromised [71]. Since 7KC increases intracellular reactive oxygen species [72–76], induces mitochondrial damage [77–79], and triggers PINK1-mediated mitophagy [13], the attenuation of *Pink1* induction by BMP enrichment suggests that BMP mitigates mitochondrial dysfunction, reducing the burden of damaged organelles requiring autophagic clearance. Additionally, BMP-mediated restoration of autophagic flux likely promotes efficient mitophagy, thereby preventing the accumulation of dysfunctional mitochondria that release pro-apoptotic factors.

Altogether, our results position BMP as an endogenous protective factor that counteracts 7KC-induced macrophage damage through integrated regulation of endolysosomal integrity and the autophagy-apoptosis crosstalk. Noteworthy, BMP accumulates in macrophages exposed to oxLDL [42,80], suggesting that endogenous BMP elevation represents an adaptive protective response that may become insufficient under chronic high-grade oxysterol exposure. Our previous studies demonstrated that BMP enrichment exerts protective effects against oxLDL-induced cellular stress by reducing oxysterol formation and inhibiting apoptosis [43,44]. Furthermore, BMP was shown to protect against cholesterol oxidation in lysosome-mimicking membrane models [46], and to serve as a reactive oxygen species scavenger in tumor cells by preventing lysosomal membrane permeabilization [81]. Hence, targeting BMP may impact the convergent mechanisms of 7KC-induced oxiapoptophagy, simultaneously restoring autophagy, preventing lysosomal damage and oxidative stress, and blocking apoptosis.

Besides atherosclerosis, other diseases such as lysosomal storage diseases (LSD) and neurodegenerative diseases are characterized by the accumulation of 7KC/oxysterols and impaired autophagy [12,14,82]. In LSD, BMP enrichment has been reported to partially compensate for reduced enzyme activity and perturbed autophagic flux [37,45,83,84]. In preliminary experiments on patient-derived NPC1-deficient fibroblasts, we observed a protective effect of BMP enrichment against 7KC-induced cytotoxicity (data not shown). With the recent characterization of enzymes regulating BMP synthesis or degradation [84–88], BMP-based interventions may emerge as new therapeutic routes for 7KC-related pathologies, in complement to emerging approaches targeting oxysterol removal [89].

## Study limitations

While this study provides novel insights into BMP's protective

mechanisms against 7KC-induced cellular damage, several limitations should be acknowledged. First, our findings are based on the murine RAW 264.7 cell line which is widely used in atherosclerosis research but may not fully recapitulate human macrophage responses to oxysterol stress. Validation in primary human macrophages would strengthen the translational relevance of these findings. Second, our study was conducted entirely *in vitro* and cannot capture the complex microenvironment of atherosclerotic plaques, including cell-cell interactions, hemodynamic forces, and chronic inflammatory stimuli. *Ex vivo* analyses would be an important next step. Third, we investigated BMP enrichment but did not perform loss-of-function experiments. Although methods for depleting BMP exist, incomplete characterization of BMP biosynthesis pathways and potential off-target effects limit the study of BMP's role in depleted cells. Fourth, while we demonstrate that BMP enrichment restores autophagic flux and prevents apoptosis, the precise molecular mechanisms linking BMP to autophagosome-lysosome fusion remain to be elucidated. Dynamic autophagy reporters could provide deeper mechanistic insights. Finally, the relationship between BMP-mediated effects on lipid homeostasis and autophagy regulation, particularly regarding lipophagy and associated enzymes, was not investigated and represents an important area for future research.

## CRediT authorship contribution statement

**Remi Lambert:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Conceptualization. **Clara Hennot:** Writing – review & editing, Validation, Methodology, Investigation, Formal analysis. **Sherine Montillet:** Writing – review & editing, Validation, Methodology, Investigation, Formal analysis. **Lars Petter Jordheim:** Writing – review & editing, Validation, Resources, Methodology. **Emeline Cros-Perrial:** Writing – review & editing, Validation, Resources, Methodology, Investigation. **Marion Guichard:** Writing – review & editing, Validation, Resources, Methodology, Investigation. **Agathe Diaz-Gonzalez:** Writing – review & editing, Validation, Methodology, Investigation, Formal analysis. **DELTON Isabelle:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization. **Celine Luquain-Costaz:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Conceptualization. **Federica Calevro:** Writing – review & editing, Validation, Resources, Methodology, Funding acquisition. **Karen Gaget:** Writing – review & editing, Validation, Resources, Methodology, Investigation, Formal analysis.

## Funding

This work was supported by INSA Lyon (Institut National des Sciences Appliquées de Lyon, France), the VML association (Vaincre les Maladies Lysosomales, France), and INRAE (Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement, France).

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

The authors gratefully acknowledge the INNOBIOVIR platform (Lyon, France) for its technical support and resources.
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