Mitophagy promotes lung repair and regeneration by restoring epithelial metabolic fitness

Domain: biology
Published: 2026-04-14
Editorial outcome: minor_revision

Abstract
Alveolar Type II cells (AT2s) are the stem cells responsible for both lung alveolar homeostasis and
regeneration. Mitochondrial dysfunction in AT2 cells has been implicated in both chronic and acute
injury-induced alveolar diseases, including idiopathic pulmonary fibrosis (IPF) and viral pneumonia.
However, the role of mitochondrial homeostasis in post-injury lung repair and regeneration remains
elusive. Here we demonstrate that genetic depletion of Ubiquitin Specific Peptidase 30 (USP30), a
negative regulator of mitophagy, boosts mitophagy and restores mitochondrial function in AT2 cells,
leading to protection from injury-induced apoptosis and enhanced stem cell activity. Both global and
AT2-specific Usp30 knockout (KO) promote alveolar regeneration, protecting the mice from bleomycin-
induced lung fibrosis and influenza pneumonia. Moreover, pharmacological inhibition of USP30
effectively alleviates these conditions. Together, our findings reveal a previously underappreciated role
for mitophagy in lung injury and repair and highlight USP30 inhibition as a promising therapeutic
strategy for treating alveolar diseases.

Introduction
Mitochondria are vital energy factories and key regulators of cellular communication, lipid metabolism,
calcium homeostasis, and apoptosis.1-10 Mitochondrial dysfunction, characterized by reduced ATP
synthesis and the accumulation of mitochondrial reactive oxygen species (mtROS), has been
implicated in numerous human diseases, including neurodegenerative diseases and respiratory
conditions like IPF and viral pneumonia.11-17 Mitophagy, the process by which damaged mitochondria
are removed and recycled, coordinates with mitochondrial dynamics and biogenesis to maintain
mitochondrial function.18, 19 Impaired mitophagy results in the accumulation of damaged mitochondria
and contributes to the pathogenesis of various diseases.

Accumulating evidence suggest that mitochondrial dysfunction and abnormalities in mitophagy
contribute to respiratory diseases. For example, IPF is a chronic and devastating lung disease caused
by repeated injuries to the alveolar epithelium, leading to abnormal or failed alveolar repair.20-25 AT2
cells, which act as stem cells in the alveoli, from patients with IPF exhibit an accumulation of damaged
mitochondria, impaired mitophagy and reduced expression of PTEN-induced kinase 1 (PINK1).26, 27
PINK1, along with Parkin, plays a crucial role in mitochondrial quality control. Pink1-/- mice show
enlarged mitochondria, defective mitochondrial function and increased susceptibility to lung fibrosis.26
Parkin deletion also exacerbates lung fibrosis in mouse model.28 The absence of mitochondrial fusion
proteins mitofusin1 (MFN1) and mitofusin2 (MFN2) in mouse AT2 cells leads to spontaneous lung
fibrosis.29 On the other hand, viral pneumonia is a leading cause of morbidity and mortality worldwide,
which may lead to acute respiratory distress syndrome (ARDS).30, 31 The severity of the infection is
often associated with mitochondrial dysfunction. For example, oxidative phosphorylation (OXPHOS)
impairment, modeled by Ndufs4 deficiency, can exacerbate influenza pathogenesis by reprograming
lung epithelial metabolism to enhance viral attachment.32 Ripk2 knockout impairs mitophagy,
enhances NLRP3 inflammasome activation, and increases mortality upon influenza virus infection.33
These findings underscore the link between these respiratory diseases and mitochondrial dysfunction
and support that restoring mitochondrial homeostasis may confer therapeutic benefits on patients.

USP30 is a universally expressed deubiquitinase that is primarily localized to the outer mitochondrial
membrane and antagonizes Parkin-mediated ubiquitination, thus functions as a key negative regulator
of mitophagy.34, 35 It preferentially removes ubiquitin chains connected via Lys6 and Lys11 linkages.35-
37 Knockout or inhibition of USP30 rescues the defective mitophagy in parkin or pink1 mutant flies,
functionally protects dopaminergic neurons in Parkinson’s disease mouse model and reduces
oxidative stress in human neurons,34, 38, 39 validating USP30 as potential therapeutic target for
Parkinson’s disease. Recently, the knockout of Usp30 has also been shown to improve heart failure
in mice caused by cardiomyocyte-specific knockout of carnitine palmitoyl transferase 2 (CPT2) by
promoting mitophagy.40 However, it is unknown whether USP30 plays a role in lung diseases.

Here, we demonstrated mitochondrial dysfunction in both IPF lungs and influenza induced pneumonia.
Using both genetic and therapeutic models, we showed that either USP30 knockout or inhibition
protects the mice from bleomycin induced lung fibrosis and influenza induced pneumonia.
Mechanistically, USP30 depletion reduces the apoptosis of AT2 cells after injury and increases AT2
cell proliferation capacity, thus restricting alveolar injury and promoting alveolar regeneration,
respectively. These data therefore support USP30 as a promising therapeutic target to treat injury
induced pulmonary diseases such as IPF and influenza pneumonia by promoting lung repair and
regeneration.

Results
Usp30 KO mice are protected from bleomycin induced lung fibrosis
A re-analysis of published transcriptomic data of pulmonary fibrosis (PF) lungs41 (GSE110147)
revealed “oxidative phosphorylation”, “aerobic respiration”, “ATP synthesis coupled electron transport”
were the top Gene Ontology (GO) terms enriched in the downregulated genes (Fig.1A, S1A),
suggesting defective mitochondrial function in PF lungs. Gene Set Enrichment Analysis (GSEA)
elucidated significant downregulation of oxidative phosphorylation and mitophagy in PF lungs (Fig. 1B
and 1C). In align with the analysis, the expression levels of PINSK1 and PRKN were negatively
correlated with fibrillar collagen expression (Fig. S1B). Re-analyzing published single-cell RNA
sequencing (scRNA-seq) data of IPF lungs42 (GSE135893) further revealed a significant
downregulation of oxidative phosphorylation in the mesenchymal, epithelial, and immune cells (Fig.
S1C and S1D). Mitophagy was significantly decreased in the epithelial cells, endothelial and immune
cells of IPF lungs, with the most pronounced changes occurring in epithelial cells (Fig. 1D). These
results collectively demonstrated that in the patients with IPF, mitochondrial dysfunction is associated
with impaired mitophagy, which correlates with the degree of fibrosis. Given mitochondrial dysfunction
has been reported to contribute to fibrogenesis,26-29 we hypothesized that enhancing mitophagy may
restore mitochondrial function and thereby inhibit lung fibrosis.

To test our hypothesis, we generated a Usp30, a gene encoding a negative regulator of PINK1 and
PARKIN-mediated mitophagy pathway,34 knockout (KO) mouse model by deleting exon 4 of the Usp30
gene. The knockout efficiency was confirmed by qRT-PCR (Fig. 1E). We further validated protein
depletion using immunofluorescence with Tyramide Signal Amplification, which confirmed reduction
of USP30 protein levels in the lung tissue (Fig. S1E). Usp30-/- mice developed and bred normally
without gross histological phenotypes, consistent with previously reported.38, 43 Bleomycin model is a
widely used lung fibrosis model. Upon bleomycin challenge, Usp30-/- mice exhibited significantly less
body weight loss compared to wild-type (WT) control mice (Fig. 1F and 1G). Masson’s trichrome
staining revealed a substantial reduction in the fibrotic areas in Usp30-/- mice (Fig. 1H and 1I). Usp30
KO significantly reduced hydroxyproline levels and fibrotic gene expression (Fig. 1J and 1K).
Immunostaining of the myofibroblast marker ACTA2 confirmed a significant decrease in myofibroblast
accumulation (Fig. 1L). These data collectively support that Usp30 KO alleviates bleomycin induced
lung fibrosis.

To examine whether Usp30 KO enhances mitophagy, we crossed Usp30 KO mice with PhAMexcised
mice, which exhibit ubiquitous expression of mito-Dendra2 fluorescence localized specifically to
mitochondria.44 By quantifying the proportion of mitochondria colocalizing with LC3B+ autophagic
puncta, we were able to quantify the mitophagy levels in mouse lungs. Our results demonstrated
Usp30 KO increased the level of mitophagy under both homeostatic conditions and following
bleomycin injury (Fig. 1M).

Usp30 deletion improves mitochondrial function and promotes alveolar regeneration
To explore the potential mechanisms by which Usp30 KO alleviates pulmonary fibrosis, we performed
scRNA-seq of the bleomycin injured lungs on day 18 post injury, analyzing both CD45+ immune cells
and CD45− non-immune cells (Fig. S1F). After rigorous quality control, we retained a total of 78,379
high-quality cells and identified 21 distinct cell types based on established marker genes (Fig. S1G
and S1H).42, 45-48 Usp30 was universally expressed in all the cell types (Fig. S1H). Changes in cell
composition between WT and Usp30 KO groups were then compared. Consistent with decreased
severity of fibrosis, Usp30 KO lungs exhibited a reduced proportion of "Proliferating" and
"Peribronchial" fibroblast clusters, which were highly enriched for the pathological fibroblast marker
Cthrc1, Postn and the myofibroblast marker Acta2 (Fig. S1I-S1K). The most pronounced change was
observed in AT2 cells, which were significantly more abundant in the KO group compared to WT group
(Fig. S1I).

AT2 cells serve as alveolar stem cells, capable of self-renewal and differentiation into mature alveolar
type 1 (AT1) cells, which are essential for gas exchange.49-51 AT2 cells are also responsible for
maintaining alveolar surface tension.52 Given the critical role of AT2 cells in maintaining alveolar
integrity and their reliance on mitochondria for energy production and survival, 49, 53 we next assessed
mitochondrial morphology, mitochondrial function and apoptosis of USP30-deficient AT2 cells.
Previous studies have reported frequent enlarged and swollen mitochondria in AT2 cells from both
IPF patients and Pink1-/- mice as shown by transmission electron microscopy (TEM) images.26 To
assess mitochondrial architecture, we employed scanning electron microscopy (SEM) and 3D
reconstruction. Under baseline conditions, the mitochondria of Usp30-KO AT2 cells displayed smaller,
more condensed cross-sectional areas compared to WT controls (Fig. S2A and S2B). Following
bleomycin injury, mitochondria in WT AT2 cells exhibited marked swelling and a globular morphology,
while Usp30-KO AT2 cells showed significantly reduced mitochondrial size and a fragmented, short
rod-like morphology. Quantitative analysis revealed that while injury induced a significant increase in
mitochondrial counts, there was no significant difference in the number of mitochondrial per cell
between WT and KO groups (Fig. S2A). Therefore, we hypothesized that Usp30 KO may enhance
mitophagy and mitochondrial biosynthesis in AT2 cells.

Next, we examined mitophagy, mitochondrial biogenesis and dynamics in the AT2 cells. The AT2
marker SFTPC was co-stained with LC3B in PhAMexcised mice, with or without Usp30 deletion. Usp30-
KO AT2 cells exhibited significantly higher levels of mitophagy compared to WT controls both under
both homeostatic conditions and following bleomycin injury (Fig. S2C), along with elevated LC3B
signal intensity (Fig. S2D). These data suggest that USP30 deletion enhances mitophagy levels in
AT2 cells.

We also examined gene expression related with mitochondrial biogenesis and dynamics using scRNA-
seq data. Usp30-KO AT2 cells displayed increased expression of Ppargc1a, the master regulator of
mitochondrial biogenesis, as well as higher expression of mitochondrial genes that encode essential
components of the mitochondrial respiratory chain (Fig. S2E). Furthermore, Usp30-KO AT2 cells
maintained higher mitochondrial OXPHOS levels while exhibiting lower glycolysis levels and reduced
mitochondrial stress (Fig. S2F), indicating a healthier metabolic profile.

To validate the transcriptome analysis, we then assessed mitochondrial function by measuring oxygen
consumption rate (OCR) in cultured WT and Usp30-/- AT2 cells. Usp30 KO significantly increased the
maximum respiration rate, basal respiration rate, and ATP production capacity under both baseline
and bleomycin-treated conditions (Fig. 2A and 2B), suggesting improved mitochondrial function. A
higher maximum respiration rate often correlates with enhanced energy production capability, thereby
improving cell viability and function.54, 55 Bleomycin induces DNA damage, thus leading to apoptosis
of the damaged epithelial cells. 56, 57 Usp30 deletion significantly inhibited bleomycin induced apoptosis
in both AT2 cultures and bleomycin injured lungs (Fig. 2C-2F). This result was further supported by
the observation of more remaining epithelial cells in the bleomycin-injured Usp30-/- lungs on day 8,
when inflammation and injury peaked (Fig. S3A).

Next, we explored the mechanism by which Usp30 deletion inhibits apoptosis. Among the numerous
deubiquitination substrates of USP30, voltage-dependent anion channel 1 (VDAC1) has been reported
as a key player of mitochondrial-dependent apoptosis.34, 58 VDAC1 forms channels on the outer
mitochondrial membrane through oligomerization, regulating the release of cytochrome c to the cytosol,
and thereby leading to mitochondrial-dependent apoptosis.59, 60 We found VDAC1 overexpression was
sufficient to increase bleomycin-induced apoptosis (Fig. S3B and S3C). In bleomycin injured lungs,
Usp30 KO decreased VDAC1 protein levels, leading to reduced apoptosis (Fig. 2F). To validate the
clinical relevance of the USP30-VDAC1 axis identified in the mouse model, we analyzed lung tissue
samples from IPF patients and healthy donors. Immunoblotting of whole lung lysates revealed
significantly elevated levels of USP30 and its substrate, VDAC1, in IPF lungs compared to healthy
controls (Fig. 2G), suggesting a conserved regulatory role of USP30 on VDAC1 protein levels in both
mouse and human lung fibrosis and also suggesting a therapeutic potential of targeting USP30 for IPF
treatment.

Epithelial injury induces apoptosis of the damaged epithelial cells, while the surviving AT2 cells
proliferate and differentiate into AT1 cells to repair the damaged regions.61-64 We then asked if AT2
stem cell activity is impacted by USP30 deletion by using organoid cultures. Interestingly, Usp30-/- AT2
organoids exhibited significantly higher colony formation efficiency (CFE) and increased proliferation
and differentiation, resulting in a significantly greater number of proliferating AT2 cells and
differentiated AT1 cells (Fig. 2H-2N). Consistent with organoid culture results, Usp30-/- mice displayed
a significantly higher rate of AT2 proliferation following bleomycin injury (Fig. 2O and 2P), which leads
to larger areas of AT2 and AT1 cells as well as a significant increase of KRT8+ intermediate cells
(DAPT/PATS/ADI),65-68 suggesting improved alveolar regeneration (Fig. 2Q, 2R, and S3D).

To further explore the molecular basis underlying increased AT2 proliferation, we assessed
mitochondrial ROS levels, mitochondrial quantity, and changes in mitochondrial membrane potential
both in vitro and in vivo. In cultured WT and Usp30-KO AT2 cells, Usp30 KO cells exhibited moderately
elevated baseline mitochondrial ROS (mtROS) and cytosolic ROS (cROS) levels, along with a
transient increase of mtROS following bleomycin injury, which then decreased, suggesting effective
mitigation of injury-induced oxidative stress (Fig. S3E-H). There were no significant differences in
mitochondria mass and mitochondrial membrane potential between WT and Usp30-KO AT2 cells at
baseline. However, following injury, mitochondria mass gradually increased until 6 hours and then
declined at 8 hours in WT cells, while KO cells exhibited an accelerated decline starting at 4 hours,
suggesting enhanced mitophagy. Consistent with this, KO cells had increased mitochondrial
membrane potential at 6 hours post injury and enhanced mitophagy flux as evidenced by P62 and
LC3-II protein level changes (Fig. S3I).

In bleomycin-injured lungs, Usp30 KO did not affect cROS levels, mitochondrial quantity nor
mitochondrial membrane potential (Fig. S3J-S3N). Instead, Usp30 KO increased mtROS levels in AT2
cells on day 8, coinciding with the increase of AT2 proliferation on day 8, followed by a rapid decline
of mtROS on day 14 during the later stage of repair (Fig. S3K). mtROS are considered as byproducts
of efficient oxidative phosphorylation,69 but it has been reported that moderate levels of ROS can
promote basal cell proliferation.70 To test if ROS levels affect AT2 cell proliferation, we introduced
hydrogen peroxide into AT2 organoid cultures to induce ROS accumulation (Fig. S3O). Lower
concentrations of hydrogen peroxide promoted AT2 cell proliferation, while higher concentration
inhibited it (Fig. S3P and S3Q). These results suggest that Usp30 KO induced a transient increase of
mtROS in AT2 cells, thereby facilitating the repair of the epithelial barrier.

In addition to AT2 cells, we explored the potential effects of Usp30 KO on other cell types, such as
macrophages and fibroblasts. Our results showed that Usp30 KO did not significantly affect
macrophage polarization (Fig. S4A and S4B). Next, to determine if the reduction in fibrosis was due
to direct effects on mesenchymal cells, we isolated primary lung fibroblasts from Usp30 KO and WT
mice. These cells exhibited comparable proliferation rates, activation, myofibroblast differentiation,
and apoptosis levels in vitro (Fig. S4C-S4H). We also examined transcriptional profiles of fibroblasts
in vivo using the scRNA-seq dataset. Usp30 deletion did not significantly affect gene expression
related to apoptosis, proliferation, or differentiation in any fibroblast subpopulation (Fig. S4I). These
data collectively suggest that the reduced fibrotic burden in Usp30 KO mice is not due to cell-
autonomous effects on fibroblasts.

In summary, our study demonstrated that Usp30 KO significantly improved mitochondrial function,
inhibited AT2 apoptosis possibly by decreasing VDAC1, and promoted AT2 stem cell activity likely by
increasing mitochondrial ROS levels, leading to alveolar regeneration and thus ameliorated lung
fibrosis.

AT2-specific Usp30 deletion effectively mitigates bleomycin injury and ameliorates pulmonary
fibrosis
Next, we asked whether AT2-specific Usp30 KO is sufficient to induce the protective effect on
pulmonary fibrosis. To explore this, we established an AT2-specific Usp30 KO mouse model,
SftpcCreERT2;R26tdT;Usp30flox/flox (hereafter Usp30ΔSftpc). Four doses of tamoxifen injections decreased
50% of USP30 expression in AT2 cells (Fig. 3A and 3B). Immunofluorescence further validated ~50%
deletion of protein specifically in lineage-labeled AT2 cells (Fig. 3C).

Subsequently, the mice were challenged with bleomycin. Compared to control group,
SftpcCreERT2;R26tdT;Usp30wt/wt (hereafter WTΔSftpc), Usp30ΔSftpc mice exhibited significantly less body
weight loss (Fig. 3D) and less severe lung fibrosis as evidenced by Masson’s trichrome staining,
quantification of fibrotic areas, hydroxyproline levels, fibrotic gene expression and ACTA2
immunofluorescence staining (Figure 3E-3H). The apoptosis levels in Usp30ΔSftpc mice were
significantly lower than those observed in the control group (Fig. 3I), while there were more
proliferating AT2 cells and a higher percentage of Sftpc-lineage-traced KRT8+ intermediate cells and
RAGE+ AT1 cells in Usp30ΔSftpc mice (Fig. 3J-3L), indicating less alveolar injury and better alveolar
regeneration, respectively. These results demonstrated AT2-specific Usp30 KO attenuates lung
fibrosis at a similar extent as global Usp30 KO. Taken together, these results established that AT2-
specific Usp30 KO is sufficient to mitigate lung fibrosis by inhibiting AT2 apoptosis and promoting AT2
proliferation.

To unravel the molecular mechanism underlying enhanced regenerative capacity, we performed
transcriptomic profiling of primary AT2 cells isolated from Sftpc-CreERT2 mice both at baseline and
post bleomycin injury. GSEA and cumulative distribution function (CDF) analysis of AT2 cells at
baseline revealed enrichment of proton motive force-driven mitochondrial ATP synthesis,
mitochondrial translation, respirasome assembly and increased mitochondrial gene expression in
Usp30-KO AT2 cells (Fig. S5A and S5B), resembling the scRNA-seq and OCR results. Pathway
analysis in Usp30-KO AT2 cells at baseline showed enrichment of Notch, Wnt, MAPK, and PI3K-AKT
signaling, which are important for stem cell activity and cell proliferation (Fig. S5C). Following
bleomycin injury, Usp30-KO AT2 cells downregulated immune responses and senescence markers
(Cdkn1a, Cdkn2a, Cdkn2b) while upregulating AT2 identity markers (Sftpc, Etv5) (Fig. S5D and S5E).
Notably, multiple genes associated with Wnt/β-catenin signaling, FGF signaling, and Hippo-YAP
signaling, which are critical for AT2 cell regeneration after injury,71-74 were upregulated, including Axin2,
Fzd1/2, Ctnnb1, Fgfr2, and Yap1. Pathway analysis showed enrichment of PI3K-AKT and MAPK
signaling (Fig. S5F). These findings suggest Usp30 KO in AT2 cells improved mitochondrial function
and upregulated signaling pathways related with stem cell activity.

Usp30 deletion confers protection against influenza pneumonia
Given Usp30 KO protects AT2 cells from apoptosis and promotes AT2 cell proliferation, we reasoned
that Usp30 deletion may also provide beneficial effects in respiratory viral infections. H1N1 influenza
A virus (IAV) induces severe respiratory epithelial damage that may lead to acute respiratory distress
syndrome and potentially progressing to lung failure.75, 76 To monitor the dynamic changes of
mitochondria specifically in AT2 cells after IAV infection, we collected the mouse lungs on multiple
time points post infection and examined mitochondrial morphology and gene expression (Fig. 4A).
Scanning electron microscopy (SEM) images demonstrated that the mitochondria in AT2 cells,
identified by the presence of their characteristic lamellar bodies (LBs), from IAV infected mouse lungs
were enlarged and swollen as early as day 2 post infection, and the number of mitochondria enclosed
by autophagic vesicles increased by day 11, indicative of undergoing mitophagy (Fig. 4B). The RNA-
seq analysis of flow-sorted AT2 cells showed that mitochondrial gene expression was suppressed at
various time points post infection (Fig. 4C), suggesting defective mitochondrial function.

The role of USP30 in IAV-induced lung injury was first investigated using Usp30-/- mice (Fig. S6A). All
Usp30-/- mice survived the influenza challenge, while half of the WT mice succumbed (Fig. S6B).
Moreover, the lung function of Usp30-/- mice, as measured by arterial oxygen saturation, declined less
and recovered more quickly (Fig. S6C). Inflammatory cell counts and protein levels in the
bronchoalveolar lavage (BAL) were comparable at early time points, however, significantly lower in
the KO mice by day 14, when the tissue injury peaked (Fig. S6D and S6E), suggesting the initial injury
was at similar levels, but there was less immune infiltration and tissue damage in the KO mice over
time. Consistently, cell apoptosis levels were significantly lower in the KO mice by day 14 compared
to the WT mice (Fig. S6F), suggesting Usp30 KO protects cells from apoptosis.

AT2 marker gene expression and alveolar epithelium covered areas were significantly higher in the
Usp30-/- mice (Fig. S6G-S6I). EdU labeling revealed a significantly higher proportion of proliferating
AT2 cells in the KO mice (Fig. S6J). We confirmed higher levels of mitophagy in the Usp30-/- mice after
influenza infection (Fig. S6K). Consistent with the results in bleomycin model, no significant changes
in total ROS levels (Fig. S6L and S6M), mitochondrial quantity (Fig. S6N) or mitochondrial membrane
potential (Fig. S6O) were observed. However, the levels of mitochondrial ROS exhibited more
pronounced dynamic changes in the KO group (Fig. S6P). Specifically, Usp30-/- mice exhibited higher
mitochondrial ROS levels on day 14, followed by a decline during the later phase of repair on day 24,
indicating a greater adaptability of the mitochondria.

Given the critical role of mitochondria in antiviral responses77, we examined whether Usp30 deletion
impacts virus control. No significant differences in viral load were observed between the WT and KO
mouse lungs (Fig. S6Q and S6R). Additionally, viral infection in AT2 organoids did not reveal any
notable differences in viral load (Fig. S6S and S6T). These findings indicate Usp30 deletion protects
the mice from influenza infection by decreasing cell apoptosis and promoting AT2 proliferation, but not
antiviral responses.

We then further validated the role of USP30 in IAV model using AT2-specific Usp30 KO mice.
Usp30ΔSftpc mice exhibited faster recovery of body weight and lung function than control group
following infection (Fig. 4D-4F). There were significantly less inflammatory cell counts and protein
levels in the BAL as well as cell apoptosis levels in the cKO mice on day 14 compared with that in
control mice (Fig. 4G-4I), indicating less tissue damage. Furthermore, AT1 and AT2 cell covered areas,
which represent epithelial barrier integrity, were restored at a higher extent in the Usp30ΔSftpc mice
compared to the control group (Fig. 4J and 4K). Sftpc mRNA levels were also higher in the Usp30ΔSftpc
group (Fig. 4L). Usp30-null AT2 cells showed a significantly higher proliferation rate (Fig. 4M). Taken
together, our results showed that Usp30 deletion has a conserved protective role across different lung
injury models by limiting AT2 cell apoptosis and promoting AT2 cell proliferation.

To investigate the molecular basis, we performed transcriptomic analysis on flow-sorted AT2 cells on
day 14 post infection. Usp30-KO AT2 cells exhibited a marked upregulation of cell cycle and mitotic
pathways, along with a downregulation of immune response-related genes, such as chemokines and
alarmins (Cxcl2, Ccl3, S100a8, Il1b), consistent with the attenuated lymphocyte activation and
recruitment observed in GSEA (Fig. S7A and S7B). Pathway analysis also highlighted enrichment in
PI3K-AKT and MAPK singaling (Fig. S7C). These transcriptomic findings validated the enhanced
proliferation of AT2 cells and improved tissue regeneration observed.

Pharmacological inhibition of USP30 ameliorates lung fibrosis and influenza pneumonia
Our results of global and conditional knockout mouse models demonstrated USP30 is a potential
therapeutic target to alleviate both chronic and acute lung injury. We thus tested this notion by using
MF094, a commercially available USP30 inhibitor that has been validated with highly selective
inhibitory effect both in vitro and in vivo.78-80 We first confirmed the inhibitory effects of MF094 on
USP30 enzyme activity and its impact on bleomycin-induced AT2 cell apoptosis (Fig. 5A). The results
showed that MF094 inhibited USP30 enzyme activity with an IC50 of 69.60 nM (Fig. 5B) and exhibited
a dose-dependent effect in suppressing bleomycin-induced apoptosis in mouse lung epithelial cell line
MLE12 (Fig. 5C). We also examined the ubiquitination of a known USP30 substrate, phospho-Ser65-
Ubiquitin (p-S65-Ub), which is phosphorylated at Ser65 by PINK1.81 MF094 treatment resulted in a
dose-dependent accumulation of p-S65-Ub following mitochondrial depolarization in MLE12 cells (Fig.
S8A), confirming effective inhibition of USP30 deubiquitinase activity.

We then tested the effects of MF094 in vivo. MF094 was intraperitoneally injected daily starting on the
third day after bleomycin injury until tissue harvest on day 14 (Fig. 5D). Mice treated with the USP30
inhibitor exhibited decreased lung fibrosis severity and less myofibroblast accumulation (Fig. 5E-5I).
The apoptotic levels were significantly lower in the inhibitor treated group (Fig. 5J).

In IAV model, MF094 was daily administrated starting from the third day post-infection till day 14 (Fig.
5K). The inhibitor treated mice exhibited less body weight loss, faster recovery of body weight and
better lung function (Fig. 5L and 5M). Inflammatory cell counts and protein levels in BAL were
significantly lower in the inhibitor group (Fig. 5N and 5O). Sftpc expression as well as AT2 and AT1
covered areas were significantly higher in the inhibitor treated group (Fig. 5P-5R), while tissue
apoptosis was significantly lower (Fig. 5S). Taken together, our studies using the two lung injury
models support USP30 as a promising therapeutic target for lung injury-induced fibrosis and viral
pneumonia.

Methods
Ethical compliance
This research complies with all relevant ethical regulations regarding animal research and human
tissue usage. All animal studies were approved by the Animal Ethics Committee of ShanghaiTech
University (protocol: 20210928001) and were performed in compliance with ethical guidelines and the
approved protocols. The human study protocol for the use of human lung tissues was approved by the
Human Ethics Committee of Shanghai Pulmonary Hospital (protocol: K21-282), and written informed
consent was obtained from all participants prior to inclusion.

Mice
Usp30-/- mice, generated by utilizing Cas9 technology to knock out a 64 bp sequence in exon 4 (gRNA1:
5'-GGCGTCCAAGACCTCGTCCT-3', gRNA2: 5'-ATGGCAGATCTCGTCCTTTG-3'), resulting in
premature termination of the encoded protein, and Usp30fl/fl mice, created by inserting flox sites at
both ends of exon 2 via Cas9 technology, allowing for deletion of exon 2 upon Cre expression, were
purchased from GemPharmatech. Sftpc-CreERT2 mice102 were generously provided by Dr. Chuwen
Lin at Sun Yat-sen University. Rosa26-tdTomato mice103 were generously provided by Dr. Harold A.
Chapman at University of California, San Francisco. PhAMexcised mice44 were generously provided by
Dr. Yifeng Zhang at ShanghaiTech University and crossed with Usp30-/- mice to examine mitophagy
levels. All experiments were carried out using mice aged 12-20 weeks, with age and sex matched,
and mice of both sexes were used in equal proportions. The mice used in this study were housed in
the Animal Core Facility at ShanghaiTech University under pathogen-free conditions, maintained at
an ambient temperature of 20-24°C and 40-60% humidity, in a 12 hours light/dark cycle room with
water and food ad libitum. All mice used in this study were on a C57BL/6 background.

Bleomycin model of lung fibrosis
After anesthetizing the mice with isoflurane, 50 µL of bleomycin (1.6 mg/kg body weight, Nippon
Kayaku) dissolved in saline was administered into the lungs via intratracheal intubation. Both control
and experimental groups consisted of sex and weight-matched mice to minimize experimental
variation. Following the model induction, mouse body weight was monitored regularly, and the mouse
lungs were collected on days 14-18.

Influenza infection
The mice were anesthetized using isoflurane to agonal breathing and intranasally administrated with
40 µL of H1N1 influenza virus PR8 (diluted in cold phosphate-buffered saline (PBS)). The viral dose
administered was approximately 36 PFU. The H1N1 influenza virus was received as a generous gift
from Dr. Haikun Wang at the Shanghai Institute of Immunology and Infection, Chinese Academy of
Sciences. Mice were weighed three times per week and euthanized at the indicated time points for
tissue harvest.

Tamoxifen treatment
Tamoxifen (Sigma-Aldrich, T5648) was dissolved in corn oil at a concentration of 40 mg/mL. To knock
out Usp30, Usp30ΔSftpc mice and the control WTΔSftpc mice were given tamoxifen at a dose of 200
mg/kg via intraperitoneal injection every other day for a total of four injections. After a 4-week waiting
period, knockout efficiency was validated, followed by experimental modeling.

Arterial blood oxygen saturation measurement
Arterial blood oxygen saturation was assessed using the MOUSEOX Pulse Oximeter system (STARR
Life Sciences). Before measurements, the thigh of the mice were shaved. During measurement, the
mice were anesthetized with isoflurane, and readings of oxygen saturation were taken using the thigh
sensor mode. After obtaining the first successful reading, measurements were conducted for at least
2 minutes per mouse, and the stable, continuous readings were averaged as the final result for each
mouse.

Lung cell isolation
The mouse lung cells were isolated based on an established protocol.104 After perfusion and lavage,
the lungs were inflated with 1 mL of pre-cold sterile Dispase II (Gibco, 17105041, 15U/mL, dissolved
in HBSS and filtered through a 0.22 µm filter) via a catheter into the trachea, followed by 0.2 mL of
warm 1% low melt agarose (BBI, A600015, dissolved in sterile ddH2O). Individual lobes were
separated, with large airways filled with agarose discarded, and the lobes were submerged with 1 mL
of Dispase II in 50 mL tubes, rocked at room temperature for 45 minutes. The tissue was chopped
using a 10 mL pipette until no visible clumps remain. Then 10 mL of DMEM (Invitrogen, 31053036)
with 2% fetal bovine serum (FBS) (Ausbian, VS500T), 1% penicillin/streptomycin (Thermo, 15140122)
and 50 U/mL DNase I (Roche, 10104159001) was added to the digested lobes, and pipetting was
performed vigorously. The mixture was swirled at 37°C for 15 minutes on a rotating platform, and the
cell suspension was filtered sequentially through 100 µm, 70 µm, and 40 µm strainers. The filtered
suspension was centrifuged for 3 minutes at 300 × g to pellet the cells. If necessary, red blood cells
were lysed by 1 mL of RBC lysis buffer (Beyotime, C3702) at room temperature for 1 minute, followed
by stopping the reaction with 5 mL of DMEM supplemented with 2% FBS and 1%
penicillin/streptomycin.

Flow cytometry
After lung tissue dissociation, cells were resuspended in DMEM supplemented with 2% FBS and 1%
penicillin/streptomycin at a density of 20 million/mL. For identifying lineage+ cells, cells were stained
with Fixable Viability Dye eFluorTM 780 (1:2000, Thermo Fisher Scientific, 65-0865-14)，FITC-
conjugated rat anti-mouse CD45 (1:1000, BD, 553080), BV711-conjugated rat anti-mouse CD31
(1:500; Biolegend, 102449), BV421-conjugated rat anti-mouse EpCAM (1:500; Biolegend, 118225),
Alexa Fluor 647-conjugated rat anti-mouse Integrin β4 (1:300; Biolegend, 123608). All cell sorting was
performed on BD FACSAria Fusion Cell Sorter. EpCAM+ cells were sorted as epithelial cells, EpCAM+
Integrin β4- cells as AT2 cells, CD45+ cells as immune cells, CD31+ cells as endothelial cells, and
EpCAM- CD31- CD45- as fibroblasts.

To assess mitochondrial-related markers in epithelial cells, BUV395-conjugated rat anti-mouse CD45
(1:300; BD, 564279), BV711-conjugated rat anti-mouse CD31 (1:500, Biolegend, 102449), BV421-
conjugated rat anti-mouse EpCAM (1:500; Biolegend, 118225), Alexa Fluor 647-conjugated rat anti-
mouse Integrin β4 (1:300; Biolegend, 123608) were used as lineage markers. Mitotracker green (200
nM; Invitrogen, M7514) was used to measure mitochondrial content, TMRM (100 nM; Invitrogen, T668)
for mitochondrial membrane potential, H2DCFDA (10 μM; MCE, HY-D0940) for cellular ROS levels,
and MitoSOX red mitochondrial superoxide indicator (5 μM; Yeasen, 40778ES50) for mitochondrial
ROS levels. Flow cytometry analysis was performed using the BD LSRFortessa X-20 and BD
FACSDiva software (version 8.0.3). Data analysis was performed using FlowJo software (version
10.4).

Bronchoalveolar lavage (BAL)
After exposing the trachea of the mouse, a 20G catheter was inserted into the trachea for lavage. One
milliliter of cold PBS was instilled into the lungs, followed by gentle aspiration, repeated three times.
All bronchoalveolar lavage fluid was collected and centrifuged at 700 x g. The supernatant was
collected to measure total protein content using the BCA assay (Thermo, 23227). The cell pellet was
lysed in red blood cell lysis buffer for one minute, the reaction was stopped with PBS, and the cells
were counted using a hemocytometer.

Hydroxyproline assay
For hydroxyproline detection, the manufacturer's protocol for the hydroxyproline kit (Sigma-Aldrich,
MAK008-1KT) was followed. Mouse lung tissues were snap frozen in liquid nitrogen and ground into
a fine powder. The powdered tissue was then hydrolyzed with 1 mL of 6 M hydrochloric acid at 120°C
for 3 hours. After cooling, the hydrolysate was neutralized with sodium hydroxide. Subsequently, 5 µL
of the hydrolysate was transferred to the center of a well in a 96-well plate. The assay reagent,
containing a chromogenic agent specific for hydroxyproline, was added to each well to facilitate a color
change. Absorbance was measured at 550 nm using a spectrophotometer, and hydroxyproline
concentrations were calculated based on a standard curve generated from known hydroxyproline
concentrations.

EdU incorporation and analysis
To detect AT2 cell proliferation after bleomycin-induced injury, 100 µg of EdU (ThermoFisher Scientific,
A10044) was administered to mice via intraperitoneal injection on day 2, 4, and 6 after injury, with lung
tissue collected on day 8. The Click-iT EdU Alexa Fluor 647 flow cytometry assay kit (ThermoFisher
Scientific, C10424) were used to examine proliferating cells via FACS. For influenza-induced injury,
proliferating AT2 cells were labeled using the same method on day 8, 10, and 12 after infection, with
lung tissue collected on day 14 for analysis.

Caspase-3/7 activity assay
After grinding the snap-frozen mouse lung tissue into a powder, protein was extracted using RIPA
buffer (50mM Tris-HCl PH7.5, 150mM NaCl, 1% deoxycholate, 0.1% SDS, 1% Triton X-100)
supplemented with Protease Inhibitor Cocktail (Roche, 11697498001), 1mM phenylmethyl sulfonyl
fluoride (PMSF) and Phosphatase Inhibitor Cocktail (Sigma Aldrich, 524625-1SET). Protein
concentration was quantified using BCA assay (Thermo, 23227). An equal amount of protein was used
to detect caspase-3/7 activity with the Caspase-Glo® 3/7 Assay (Promega, G8091). Briefly, 5 µg of
total protein was diluted in PBS to a volume of 40 µL, then mixed with 40 µL of the reaction solution in
a clear-bottom 96-well plate. The mixture was shaken at 300-500 rpm for 5 minutes and incubated at
room temperature for 30 minutes. The solution was then transferred to a white opaque 96-well plate
and measured using a Spark Multimode Microplate Reader (Tecan, Switzerland). Two replicates were
set up for each sample, with the average of the two replicates as the final reading.

Masson’s trichrome staining
Trichrome stain was performed on the frozen lung sections according to the standard procedure of
Masson Stain Kit (Yeasen, 60532ES74) with modifications. Briefly, OCT embedded frozen sections
were equilibrated to room temperature and rehydrated in distilled water for 1 minute before
sequentially stained with hematoxylin for 1 minute, acidic fuchsin for 2 minutes, phosphomolybdic acid
for 5 minutes, and aniline blue for 3 minutes, with three washes with washing solution between each
staining step. After the final dehydration step in anhydrous ethanol, the sections were mounted for
microscopic analysis.

For quantitative assessment of fibrosis, stained sections were analyzed using Fiji/ImageJ software.
The color deconvolution plugin was first applied using the Masson Trichrome vector set to isolate the
collagen-specific blue channel. The threshold function was then used to select and quantify collagen-
positive areas, while total lung area was determined using the WEKA Trainable Segmentation plugin
for 2D image analysis. The percentage of fibrotic areas was calculated as the percentage of collagen-
positive areas relative to total lung areas.

AT2 organoid culture
Mouse AT2 cells were flow sorted from WT or Usp30-/- mouse lungs, resuspended at 3,000 cells per
40 μL droplet in a 1:1 mixture of AT2 Maintenance Medium (AMM)105 (without CHIR99021 and IL-1β)
and Matrigel® (Corning, 356231), plated in 24-well plates, and allowed to solidify at 37°C for 30 min
before adding 500 μL pre-warmed AMM (without CHIR99021 and IL-1β). Medium was changed every
three days. Samples were collected after 10 days of culture. For oxidative stress experiments,
organoids were treated with H2O2 (Sinopharm, 10011208) at various concentrations of 1 μM, 5 μM, or
10 μM in fresh AMM (without CHIR99021 and IL-1β) throughout the entire duration of the experiment.

To analyze the colony formation efficiency, each droplet was imaged using a Nikon TI-E microscope
in bright-field mode. Z-stack images were combined, and the number of organoids was counted using
ImageJ. In ImageJ, the image contrast was enhanced using the "Subtract Background" function. A
threshold was applied to distinguish colonies from the background. If colonies appeared clumped, the
watershed function was used to separate them. Finally, colonies were counted using the "Analyze
Particles" function, with appropriate size settings to exclude noise and adjustments to the circularity
parameter. Only organoids with more than five nuclei were included.

Influenza infection of AT2 organoids
For viral infection, AT2 organoids were initially cultured at a density of 5,000 cells in the complete AMM
medium for 10 days. Then, the droplets were digested with Dispase II (15 U/mL ; Gibco, 17105041)
at 37°C for 30-40 minutes to be released from Matrigel. One to two droplets from each group were
further digested with TrypLETM (ThermoFisher Scientific, 12604013) for 5-10 minutes to obtain single
cells for cell count reference. The rest of organoids were centrifuged, washed with PBS and then
mechanically sheared using 1 mL pipette tips to expose the apical surface for viral infection. The virus
was added at a multiplicity of infection (MOI) of 0.01 and incubated at 37°C for 1 hour. After infection,
the organoids were washed three times with PBS, reseeded in 40 µL of a 1:1 Matrigel:Medium mixture
in a 24-well plate at a density of 100,000 cells per well. Organoids were collected at various time points,
washed three times with PBS followed by snap frozen in liquid nitrogen for RNA extraction to measure
the viral load.

Oxygen consumption rate (OCR) measurement
Cells from AT2 organoids after 10-d culture were digested using Dispase II and TrypLETM as above
mentioned and seeded for OCTR measurement. Prior to cell plating, each well of the Seahorse XF96
plate (Aligent) was coated with Matrigel at 1:20 dilution in PBS and incubated for 2 hours at 37°C. AT2
cells were then seeded at a density of 30,000 cells per well in the complete AMM medium and allowed
to attach for 24 hours in a humidified incubator at 37°C with 5% CO₂. Eight hours prior to OCR
measurement, cells were treated with 1 mg/mL bleomycin (Vokai Biotechnology, 9041-93-4) or vehicle
control.

The Seahorse assay was performed according to the manufacturer’s protocol. The culture medium
was replaced with Seahorse XF assay medium supplemented with appropriate substrates. Plates
were equilibrated at 37°C without CO₂ for 1 hour before OCR measurements. The OCR was measured
under basal conditions and after the sequential addition of inhibitors (1.5 μM oligomycin, 1 μM FCCP,
and 0.5 μM each of rotenone/antimycin A) to assess mitochondrial function. Each measurement
included three baseline readings followed by injections of oligomycin and FCCP to evaluate ATP
production and maximal respiratory capacity, respectively. Data were analyzed using Seahorse Wave
software, and OCR was expressed in pmol/min.

In vitro mitochondrial function and mitophagy flux assay
Cells from AT2 organoids were dissociated as described above and seeded in 12-well plates pre-
coated with Matrigel (1:20 diluted) at a density of 250,000 cells per well. After 24 hours, cells were
treated with bleomycin (2 mg/mL; Vokai Biotechnology, 9041-93-4) or vehicle control. For flow
cytometric analysis, cells were digested with 0.25% trypsin at indicated time points. Mitochondrial
parameters were assessed using Mitotracker green (200 nM; Invitrogen, M7514), TMRM (100 nM;
Invitrogen, T668), H2DCFDA (10 μM; MCE, HY-D0940), and MitoSOX red (5 μM; Yeasen,
40778ES50). Data acquisition was performed on a BD LSRFortessa X-20. For mitophagy flux analysis,
cells were treated with bleomycin (2 mg/mL) or vehicle for 4 hours. Bafilomycin A1 (100 nM) was
added 2 hours prior to harvest, followed by protein extraction and Western blot analysis.

Cell line culture
MLE12 cells (ATCC, CRL-2110) were cultured in HITES medium (Dulbecco's:Ham's F12, 50:50 mix
supplemented with 2% FBS (Ausbian, VS500T), 0.005 mg/mL insulin (Tocris, 3435/10), 0.01 mg/mL
transferrin (Roche, 10652202001), 30 nM sodium selenite (Sigma-Aldrich, S9133), 10 nM
hydrocortisone (MCE, HY-N0583), 10 nM β-estradiol (Sigma-Aldrich, E2758), and 2 mM L-glutamine
(Solarbio, G0200) at 37°C with 5% CO₂. HEK293T cells (ATCC, SNL-015) were cultured in DMEM
(Invitrogen, 31053036) supplemented with 10% FBS (Ausbian, VS500T) at 37°C with 5% CO₂. Both
cell lines were authenticated by STR profiling by the supplier and were routinely tested to ensure they
were free of mycoplasma contamination.

Plasmid
The full-length human VDAC1 coding sequence was amplified from human cDNA and cloned into the
pcDNA3.1(+) vector to construct the pcDNA3.1-VDAC1 plasmid. All newly generated plasmids are
available from the corresponding author upon request.

USP30 inhibitor
The MF094 inhibitor (MCE, HY-112438) was dissolved in dimethyl sulfoxide (DMSO) to create a stock
solution of 125 mg/mL. Prior to administration, the stock solution was diluted 10-fold with corn oil
(Abmole, M9109). Daily intraperitoneal injections of the diluted solution were administered to mice at
a dose of 30 mg/kg.

Fluorescence-based USP30 enzyme activity assay
Recombinant human USP30 (rhUSP30; R&D Systems, #E-582) was diluted to 25 nM in assay buffer
(50 mM Tris pH 7.5, 100 mM NaCl, 0.1 mg/mL BSA, 0.05% Tween-20, 1 mM DTT) and 5 µL was
added to each well of black, clear-bottom 384-well plates (PE, #6008280). Two point five µL of MF094
was added in each well. After 30 min pre-incubation at room temperature, 2.5 µL of Ub-Rho110
substrate (R&D Systems, #U-555; final 50 nM) was added to initiate the reaction (final volume: 10 µL).
The plate was incubated for 1-2 hours at room temperature, with inhibitor-only and enzyme-only
controls included. Fluorescence was measured at 485 nm excitation and 540 nm emission.

Cell-based apoptosis assessment
Cells from AT2 organoids were digested using Dispase II and TrypLETM as above mentioned and
seeded for apoptosis assay at a density of 50,000 cells per well in a 96-well plate which has been pre-
coated with a 1:20 diluted Matrigel for 2 hours. MLE12 cells were seeded at 20,000 cells/well in a 96-
well plate and pretreated with MF094 for 2 hours. HEK293T cells were seeded at 20,000 cells/well in
a 96-well plate and transfected with 200 ng pcDNA3.1-VDAC1 plasmid using Lipofectamine 2000
(Invitrogen, 11668) 24 hours before the following assay.

For apoptosis detection, the cells were treated with 1 mg/mL bleomycin (Vokai Biotechnology, 9041-
93-4), incubated with IncuCyte® Caspase-3/7 Green (Sartorius, 4440) at 1:1000 dilution and
monitored by IncuCyte® live cell system in real time. The ratio of cells undergoing apoptosis was
calculated as the number of green fluorescent cells divided by the total cell areas.

Bone marrow derived macrophage (BMDM) culture
Both WT and Usp30-/- BMDMs were isolated and cultured as follows: mouse femurs and tibias were
ground and the bone marrow was flushed with sterile PBS, filtered through a 40 µm cell strainer and
centrifuged. The cell pellet was resuspended in RPMI-1640 medium (Invitrogen, C11875500CP)
supplemented with 10% FBS (Ausbian, VS500T), 1% penicillin/streptomycin (Thermo, 15140122), and
1% HEPES (Invitrogen, 15630080), plated at 8 million cells per 10 cm non-treated dish and incubated
at 37°C in a 5% CO₂ atmosphere. On day 4, 5 mL of fresh medium was added. On day 7, cells were
digested with 2 mM EDTA for 10 minutes at 4°C. The macrophages were collected and plated at
800,000 cells per well in a 12-well plate. For polarization, cells were stimulated with LPS (100 ng/mL;
InvivoGen, tlrl-eklps) and IFN-γ (20 ng/mL; R&D, 485-MI-100) for M1 polarization, or IL-4 (20 ng/mL;
PeproTech, 214-14) for M2 polarization. After 24 hours, cells were collected for RNA extraction and
qPCR analysis.

Lung fibroblast culture
Both WT and Usp30-/- fibroblasts were isolated from mouse lungs by flow sort based on EpCAM-CD31-
CD45-, as detailed in the flow cytometry section. The flow-sorted fibroblasts were cultured in DMEM
(Invitrogen, 31053036) supplemented with 10% FBS (Ausbian, VS500T) and 1%
penicillin/streptomycin (Thermo, 15140122) and used between passages 2 and 5. For fibroblast
activation assays, WT and Usp30-/- fibroblasts were seeded in a 12-well plate at the density of 50,000
per well for RNA extraction, or in 8-well chamber slides at a density of 200,000 cells per well for
immunofluorescence. Cells were stimulated with 4 ng/mL of TGF-β1 (R&D, 240-B-010) for 48 hours,
followed by RNA extraction for qRT-PCR or fixation and staining. To evaluate fibroblast proliferation,
WT and Usp30-/- fibroblasts were seeded in a 96-well plate or in 8-well chamber slides at the density
of 20,000 per well and cultured with or without 10% FBS (Invitrogen, 31053036) for 48 hours followed
by CellTiter-Glo assay (Promega, G7571) or EdU analysis. For EdU analysis, 10 µM EdU was added
4 hours prior to harvest, followed by staining as described in the EdU incorporation section. For
apoptosis assessment, fibroblasts were seeded in 96-well plates at a density of 20,000 cells per well.
The following day, cells were changed to serum-free medium and cultured for 48 hours followed by
caspase-3/7 activity determination using IncuCyte® Caspase-3/7 Green (Sartorius, 4440) as
described in the cell-based apoptosis assessment section.

Scanning Electron Microscopy (SEM)
Freshly collected lung samples were fixed in 2.5% glutaraldehyde in PBS at room temperature for 2
hours, followed by PBS wash and post-fixation in 1% osmium tetroxide in PBS for 1 hour. After that,
the samples were dehydrated through a graded series of ethanol (30%, 50%, 70%, 90%, and 100%)
and embedded in epoxy resin. Ultrathin sections (70 nm) were cut using a Leica Ultracut UCT
microtome and collected on copper grids. The sections were then stained with 2% uranyl acetate for
15 minutes and lead citrate for 5 minutes to enhance contrast. SEM imaging was performed using a
GeminiSEM460 Field Emission Scanning Electron Microscope, with a minimum of 20 cells per sample
examined to ensure representative imaging.

To quantify mitochondrial morphological parameters and perform 3D reconstruction, SEM images
were analyzed using a customized computational pipeline. Mitochondrial segmentation was performed
using Cellpose v4106 with a CPSAM model that was pre-trained on mitochondrial datasets and
subsequently fine-tuned through multiple rounds of iterative training (≥10 rounds) using manually
annotated SEM images from our samples until achieving a segmentation accuracy exceeding 90%.
The resulting binary masks were analyzed in ImageJ (Fiji) using the Analyze Particles function to
quantify mitochondrial counts and cross-sectional areas. For 3D reconstruction, serial ultrathin
sections (70 nm thickness, n ≥ 100 consecutive sections per sample) were first aligned and registered
in TrakEM2 (Fiji) to correct for cutting-induced distortions, followed by mitochondrial segmentation
using the optimized Cellpose model. Aligned segmentation masks were subsequently imported into
Amira software (v6.0, Thermo Fisher Scientific), where the following steps were performed
sequentially: interactive thresholding, surface generation, surface smoothing, and surface visualization.
This workflow enabled precise assessment of mitochondrial morphology and spatial structure.

Tissue preparation and immunofluorescence (IF)
The freshly dissected mouse left lungs were inflated with 600 μL fixation solution (4% PFA, 50% OCT)
and fixed at room temperature for 1 hour, followed by PBS wash, overnight dehydration in 15%
sucrose and 50% OCT and subsequently OCT embedding. The tissue block was sectioned into 6 μm
slices using a Cryostat Microtome (ThermoFisher Scientific). The sections were fixed with 4% PFA for
5 minutes, treated with 0.1% sodium borohydride (Sigma Aldrich, 213462) in PBS for 10 minutes,
blocked for 1 hour in blocking buffer (PBS supplemented with 1% BSA, 5% horse serum and 0.1%
Triton X-100), and incubated with the primary antibody at 4°C overnight. The next day, the sections
were stained with a fluorescent secondary antibody for 1 hour at room temperature, followed by DAPI
staining for 15 minutes, and then mounted for imaging.

For Tyramide Signal Amplification (TSA) staining, the tissue sections were processed using a TSA kit
(ABclonal, RK50026P) with modifications to the standard immunofluorescence protocol described
above. Following sodium borohydride treatment, endogenous peroxidase activity was quenched by
incubation with Peroxidase Blocking Buffer for 15 minutes at room temperature in the dark. After three
washes with PBS, blocking and primary antibody incubation were performed as previously described.
The sections were then incubated with HRP-conjugated rabbit polymer secondary antibody (1:1000)
for 1 hour at room temperature. Immunofluorescence signals were developed by incubation with the
fluorescent tyramide working solution for 10 minutes at room temperature. Nuclei were counterstained
with DAPI for 15 minutes, and the sections were mounted for imaging.

The antibodies used in this study included rabbit anti-pro-SPC (1:3000; Millipore, AB3786), goat anti-
pro-SPC (1:1000; Santa Cruz Biotechnology, sc-7706), mouse anti-pro-SPC (1:50; servicebio,
GB154201), rat anti-RAGE (1:400; R&D, MAB1179-100), rabbit anti-phospho-Histone H3(Ser10)
(1:500; Sigma-Aldrich, 06-570), rabbit anti-LC3B (1:100; Sigma Aldrich, L7543), mouse anti-ACTA2-
conjugated FITC (1:200; Sigma Aldrich, F3777), rabbit anti-ACTA2 (1:500; Abcam, ab5694), rat anti-
KRT8 (1:100; DSHB, MABT329M) and rabbit anti-USP30 (1:50; ThermoFisher Scientific, PA5106762).

Quantification of immunofluorescence
For quantification of immunofluorescence staining of tissue sections, the entire lung lobes were
imaged using the Nikon Ti2-E CSUC-W1 Sora camera system (Nikon, Japan), except that Cy7 signals
identifying mitochondrial autophagy in AT2 cells were acquired using the STELLARIS 8 Falcon FLIM
Confocal microscope (Leica Microsystems, Germany). The Trainable Weka Segmentation (TWS)
plugin in Fiji/ImageJ was employed to quantify the areas of ACTA2+, RAGE+, and their corresponding
DAPI+ regions. SPC+ areas were manually outlined in ImageJ by selecting regions with homogeneous
SPC distribution. For quantification of organoid cultures, SPC+, RAGE+, phospho-Histone H3+, and
EdU+ cells were quantified using QuPath v0.5.1 with “Analyze → Cell Detection → Positive Cell
Detection” module. For the quantification of mitophagy, QuPath v0.5.1 software and module were
employed. Each mitochondrion was identified based on the mito-Dendra2 signal. The colocalization
of LC3B with each mitochondrion was used to determine the mitochondrion undergoing mitophagy,
and mitophagy index was calculated as the ratio of LC3B+mito-Dendra2+ to total mito-Dendra2+
mitochondria.

Western blot
Snap-frozen mouse lungs were ground into powder on dry ice and lysed in RIPA buffer supplemented
with Protease Inhibitor Cocktail (Roche, 11697498001), 1mM phenylmethyl sulfonyl fluoride (PMSF)
and Phosphatase Inhibitor Cocktail (Sigma Aldrich, 524625-1SET). Protein concentration was
measured using the Pierce BCA protein assay kit (ThermoFisher Scientific, 23227) and equal amounts
of protein lysates were loaded for SDS-Page. The primary antibodies employed were β-Actin (1:1000;
Santa Cruz Biotechnology, sc-47778), VDAC1 (1:400; Santa Cruz Biotechnology, sc-390996),
Cleaved caspase-3 (1:1000; Cell Signaling Technology, 9661S), USP30 (1:500; ThermoFisher
Scientific, PA5106762), COL1A1 (1:1000; Cell Signaling Technology, 72026S), ACTA2 (1:200; Abcam,
ab5694), LC3B (1:500; MBL M152-3), P62 (1:500; Santa Cruz, sc-48402), Phospho-Ser65-Ubiquitin
(1:1000; Cell Signaling Technology, 62802S) and pro-SPC (1:400; Millipore, AB3786). The secondary
antibodies used were goat anti-mouse conjugated HRP (1:5000; Thermo Fisher Scientific, G21040)
and goat anti-rabbit conjugated HRP (1:5000; Thermo Fisher Scientific, G21234).

Measurement of Phospho-Ser65-Ubiquitin levels
MLE12 cells were seeded in 12-well plates at a density of 200,000 cells per well. Cells were pretreated
with MF094 at concentrations of 0, 1, 5, 10, and 20 µM for 2 hours prior to the addition of 10 µM CCCP
for 4 hours. Cells were harvested and the protein lysates were prepared for Western blot analysis.

RNA extraction and qPCR analysis
For RNA extraction, the Eastep™ Super Total RNA Extraction Kit (Promega, LS1040) was used for
tissue samples, while the HiPure Total RNA Nano Kit (Magen, R4125) was employed for cell samples.
Complementary DNA (cDNA) was synthesized using a reverse transcription kit (Vazyme, R222-01).
Subsequent qPCR analysis was conducted with SYBR qPCR Master Mix (Vazyme, Q711-03).
Relative gene expression levels were determined using the ΔΔCT method and normalized to HPRT.
The primers utilized in this study are listed in Supplementary Table 1.

RNA-seq analysis
AT2 cells (CD45- EPCAM+ CD31- Integrin β4-) were flow-sorted from the lungs of influenza-infected
mice at 0, 2, 6, and 11 days post-infection. Sftpc-traced AT2 cells (tdTomato+) were flow-sorted from
tamoxifen-treated WTΔSftpc and Usp30ΔSftpc mice at day 14 after saline or bleomycin administration, or
at day 14 after influenza infection. Following RNA extraction, library preparation was performed using
the TruSeq RNA sample preparation kit (Illumina, RS-122-2102) according to the manufacturer's
protocol. Sequencing was conducted on the Illumina NovaSeq 6000 platform, generating 150 bp
paired-end reads. Raw sequencing data were processed using bcl2fastq (Illumina) to convert base
call files to FASTQ format. Quality control was performed using FastQC (V0.12.1) to assess the
FASTQ files.107 Reads were aligned to the Mouse Genome Assembly GRCm38 using HISAT2
(V2.2.1).108 The resulting alignment files were processed to generate gene expression counts using
featureCounts (V1.22.2).109 For the time-course samples, differential expression analysis was
conducted using edgeR (V3.40.2).110 For Sftpc-traced AT2 cell datasets (saline, bleomycin, and
influenza groups), differential expression analysis was performed using DESeq2 (V1.38.3).111 Gene
set enrichment analysis (GSEA)112 and KEGG pathway analysis were performed using
clusterProfiler113 (V4.1.2). For saline-treated Sftpc-traced AT2 cells, the log fold changes of
mitochondrial genes were compared to those of background genes (non-mitochondrial genes).
Cumulative distribution function (CDF) curves were generated using the ggplot2 package (V3.4.4) in
R, and the statistical significance of the distribution shift was assessed using the Kolmogorov-Smirnov
(KS) test.

For re-analysis of published microarray data (GSE110147),41 raw CEL files were processed using the
R packages affy114 (V1.76.0) and limma115 (V3.54.0), with differentially expressed genes defined as
|log FC| ≥ 1 and p value ≤ 0.05. GSEA and correlation analyses were performed using
clusterProfiler113 (V4.1.2) and cor.test function, respectively.

Single cell RNA-seq analysis
CD45+ and CD45- cells were flow-sorted from bleomycin treated WT and USP30-/- mice (n= 4 mice /
group) and processed using the Chromium Single Cell Platform (10X Genomics, Pleasanton, CA).
Libraries were prepared according to the manufacturer's protocol and sequenced on an Illumina
NovaSeq 6000 system. The CD45+ and CD45- library yielded 79,493 cells with an average sequencing
depth of 22,802 reads per cell. Raw sequencing data were processed using Cell Ranger v8.0.1 (10X
Genomics) for initial quality control. Subsequent analyses were performed using the Seurat
package116 (v4.3.0) in R. We applied stringent quality filters, excluding cells with fewer than 200
detected genes or >25% mitochondrial gene content. After quality control, 78,379 high-quality cells
were retained for downstream analysis.

Data normalization and feature selection were performed using SCTransform. Dimensionality
reduction was achieved through principal component analysis (PCA), with the top 20 principal
components serving as input for UMAP visualization. Cell clustering was performed using the
FindClusters function (resolution = 0.8), and cell type annotation was based on canonical marker gene
expression patterns. To evaluate specific cellular phenotypes and metabolic states, pathway activity
scores were computed using the AddModuleScore function in Seurat with default parameters. For
fibroblasts, functional states were assessed using gene sets for proliferation (Mki67, Pcna, Top2a,
Ccna2, Cdk1, Ccnb1), apoptosis (Bax, Casp3, Casp8, Fas, Bid, Bcl2l11), and differentiation (Acta2,
Col1a1, Fn1, Postn, Cthrc1, Spp1). For AT2 cells, metabolic profiles were characterized using gene
sets for glycolysis (Pkm, Ldha, Gapdh, Hk2, Eno1, Pgk1, Pfkp), mitochondrial stress (Hspd1, Hspa9,
Clpp, Lonp1, Yme1l1), nuclear-encoded OXPHOS (Ndufa9, Ndufb8, Sdhb, Uqcrc2, Atp5a1, Cox5a,
Cox4i1), and mitochondria-encoded OXPHOS (mt-Nd1, mt-Nd2, mt-Co1, mt-Co2, mt-Atp6, mt-Cytb).

The published scRNA-seq data of IPF lungs (GSE135893)42 was reanalyzed using the Seurat116
(V4.3.0) package in R (V4.2.2). Differential gene expression analysis between IPF and control samples
was performed using the FindMarkers function with default parameters, followed by gene set
enrichment analysis (GSEA)112 to identify significantly altered pathways.To assess mitophagy and
oxidative phosphorylation activity scores, we retrieved the corresponding gene sets from the Gene
Ontology database (GO:0000423 for mitophagy and GO:0006119 for oxidative phosphorylation).
Cellular pathway activity scores were calculated using Seurat’s AddModuleScore function116 (V4.3.0)
with default parameters, enabling quantitative comparison across cell populations.

Human sample collection
Human lung tissues were obtained from Tongji University Affiliated Shanghai Pulmonary Hospital.
Specifically, normal lung tissues were obtained from macroscopically normal tissues adjacent to
pulmonary nodules during surgical resection, and IPF lung tissues were obtained from the explanted
lungs of IPF patients undergoing lung transplantation. Participants were not compensated. To protect
participant anonymity, exact ages have been replaced with age ranges in our data presentation.
Following the SAGER guidelines, participant sex (3 females and 7 males) was determined based on
self-reported medical records. Due to the limited sample size used exclusively for immunoblotting,
sex-based analysis was not performed. All human tissue samples were completely destroyed during
the protein extraction process for immunoblotting analysis, and no samples were conserved for further
analysis. Detailed de-identified characteristics of the human tissues are listed in Supplementary Table
2.

Statistics and Reproducibility
Custom scripts in R v4.2.2 were utilized for RNA-seq and scRNA-seq data analysis and plotting. All
other statistical calculations were analyzed using GraphPad Prism version 9.0 (GraphPad Software,
San Diego, CA). Statistical tests used and the sample size is indicated in corresponding figure legends.
The exact p-values are shown in figures or figure legends. A p value of <0.05 was considered
statistically significant. All data are generated from three or more independent experiments with at
least three biological replicates per genotype and condition. Investigators were blinded to group
allocation during the quantitative assessment of imaging analysis and lung function monitoring. For
biochemical assays and computational sequencing analysis, blinding was not applicable as group
identity is required for downstream comparative pipelines. No statistical methods were used to
predetermine sample sizes. Sample sizes were empirically chosen based on established standards in
pulmonary research, which provide sufficient statistical power to detect biological differences in injury
models.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript identifies USP30 as a novel therapeutic target in lung injury. The authors employ both global and AT2-specific genetic knockout models, providing compelling evidence that USP30 inhibition promotes lung regeneration in diverse injury contexts, including bleomycin-induced fibrosis and influenza-induced pneumonia. Mechanistically, USP30 depletion reduces AT2 cell apoptosis via VDAC1 following injury while enhancing their proliferative capacity, thereby limiting alveolar damage and promoting regeneration. Overall, the study provides important insights into USP30 as a regulator of alveolar repair and highlights mitochondrial dysfunction as a promising therapeutic avenue, but several points require clarification and further analysis.\nMajor Points:\n1. Clarify whether USP30 deletion affects mesenchymal/fibroblast populations (apoptosis, proliferation, or differentiation) and whether fibroblasts show transcriptional changes in scRNA-seq.\n2. Assess whether mitochondrial functions beyond mitophagy (e.g., oxidative phosphorylation) are altered in scRNA-seq data.\n3. Determine if USP30 is expressed in transient cell states (DAPT/PATS/K8+) and whether knockout in AT2 cells impacts these populations post-injury.\n4. Evaluate potential effects of USP30 deletion on distal bronchiolar stem/progenitor cells (secretory cells, BASCs, LNEPs).\n5. Provide clarification on the design of the floxed allele and possible reasons for limited recombination efficiency in AT2-specific KO mice (Figure S3B). Could this be related to the sequence length between two loxP sites in the floxed USP30 allele, making excision less efficient even with the robust Sftpc-CreERT2 driver?\n6. Include lineage-tracing data (tdTomato) in AT2-specific KO experiments (Figure3) to better distinguish AT2-derived regeneration from bronchiolar stem cell contributions. More precise quantification of AT2 proliferation (tdT+ vs tdT–) is needed.\nMinor Points:\n1. The statement that USP30 KO “does not affect” fibroblasts or macrophages may be too strong according to limited data here; consider rephrasing.", "reviewer_2": "Wu et al. investigate the role of USP30-mediated mitophagy in promoting alveolar regeneration following lung injury. The authors used genetic and pharmacological approaches targeting Usp30 to demonstrate that loss of USP30 enhances mitophagy and restores mitochondrial function in AT2 cells, thereby protecting them from apoptosis and promoting regenerative capacity. The findings are interesting and provide a fresh insight into the cellular mechanisms of tissue repairing. The study nicely shows that Usp30 deletion reduces AT2 apoptosis and promotes proliferation across multiple lung injury models. It can be further strengthened to examine the link between USP30 loss and changes in the AT2 cell transcriptomic profile with or without injury, providing evidence how USP30 loss enhances AT2 stem cell activity at the molecular level. A few other minor issues are listed below.\n1. The authors performed scRNA-seq for Usp30-/- and control mice followed by bleomycin challenge. It is interesting that no pathological fibroblasts were identified. This reviewer wonder if the authors examine pathological fibroblasts markers Cthrc1 and Postn?\n2. Figures S1I and S1J: Saline-treated controls can be further analyzed. Data on mitochondrial number and function in uninjured lungs would strengthen the baseline comparison.\n3. Figures 3A–3C: It is unclear whether observed mitochondrial morphology and gene expression changes are specifically from AT2 cells. Additional validation such as AT2-specific isolation or imaging would clarify this point.\n4. Figure 3C heatmap and KEGG analysis: The comparison focuses on time points rather than genotypes. It would be more informative to directly compare WT and Usp30⁻/⁻ gene expression profiles to identify pathways altered by USP30 loss.\n5. The authors cultured AT2 organoids in AMM-based medium as described in Ref 91. It seems that AT1 cell differentiation rarely happens if cultured in AMM. It is interesting that the authors detected quite a number of AT1 cells in this culture system (Fig. 2L, M). Did the authors modify the medium?", "reviewer_3": "Studies by Wu et al, analyzes the susceptibility to lung injury and fibrosis by inhibition of USP30 using genetic and pharmacological approaches. Studies analyze the bleomycin induced lung fibrosis and the influenza pneumonia murine models. Studies show that inhibition of USP30 in AT2 cells is sufficient to increase resilience against lung injury. Investigators hypothesize inhibition of USP30 promotes AT2 proliferation by an ROS mediated mechanisms and protection against apoptosis by reducing VDAC protein levels. The studies are relevant to the field and have translational potential. However, there are some concerns that need to be addressed.\n1. The significance of the studies, especially the translational potential, will be enhanced with evidence of higher expression of USP30 in AT2 cells of human lung fibrotic diseases like IPF. Similarly, if changes in ubiquitination and protein levels of VDAC1observed in the current mouse studies are relevant for the human fibrotic disease.\n2. scRNAseq data do not align with the other set of data of the manuscript. For example, the proportion of myofibroblasts that the authors show increase by IFA are not shown, nor the cluster of fibrotic fibroblasts CTHRC1+ that produce high levels of ECM. These changes will confirm the effect in fibrosis by the absence of USP30. Similarly, it would be important to define the cluster of proliferating AT2 to confirm the EdU and organoid experiments. Additionally, there is lack of AT1 cell cluster. The AT2-AT1 cluster shown in the UMAP data has a limited proportion in comparison with the abundant Ager+ staining shown in Figure 2.\n3. Interestingly, studies in Figure 2 and 2S show that AT2 cells of WT mice in naïve conditions have active apoptosis in contrast to AT2 from USP30 KO. Authors suggest that this apoptosis is related to high levels of VDAC protein. It is unclear what is the stimuli for overexpression of VDAC1 in naïve healthy AT2 cells to mediate apoptosis. Furthermore, transcriptomic data is needed to demonstrate that changes in VDAC in USP30 deficient AT2 cells are not associated with transcriptional changes and only are related to changes in ubiquitination and protein degradation.\n4. The high levels of mtROS in AT2 from USP30 KO mouse are in contrast to the increase in maximal respiration shown in the seahorse studies for KO cells. High maximal OCR generally is associated with a state of low mtROS generation and mitochondrial fitness. Furthermore, it is unclear why there are not changes in the mitochondrial TMRM and mitochondrial mass between mouse strains if mitophagy is induced differently between groups. Simultaneous analysis of these two parameters might help to give a better understanding as well as complementary markers of autophagy like p62 protein levels, and ubiquitination of Parkin to analyze mitophagy. Furthermore, studies of mitophagy flux in the AT2 cells might be also useful.\n5. Figure 1M do not show markers of cell type to define if AT2 cells have been analyzed for the colocalization of mitochondria and LC3B. it seems that USP30 deficiency increase levels of LC3B.\n6. The authors in line 162 suggest enhanced mitochondrial biosynthesis in AT2 from USP30 KO suggested by mitochondrial morphology and number. Complementary studies of markers of mitochondrial biogenesis and dynamics are recommended to support the statement.\n7. Figure 4B shows inhibition rate of the Compound MF094, however it will be more specific to show the effect of the compound on the ubiquitination of a known mitochondrial protein target of USP30.\nMinor concerns\n1. The measurements for OH-Pro for bleomycin and influenza pneumonia lung models have more data points than other experimental variables, suggesting than more than 1 sample per lung mouse was analyzed. There is not a description in this respect on methods. Please clarify.\n2. The USP30 protein expression and levels after deletion were not analyzed"}, "author_response": "Response to the reviewers' comments:\nWe thank the reviewers and the editors for their efforts in evaluating our manuscript and\nproviding very constructive suggestions. We have performed serials of experiments to address\nall the reviewers' concerns/questions.\nIn particular, we provide additional evidence to: (1) investigate mitochondrial function by\ncomprehensive transcriptomic analysis and multi-timepoint dynamic assessment; (2) validate\nthe experimental findings by in-depth transcriptomic analysis; (3) perform additional RNA-seq\non AT2-specific Usp30 KO mice to investigate the molecular mechanism; (4) assess AT2-\nspecific mitophagy; (5) determine USP30 and VDAC1 protein levels in clinical specimens; (6)\nvalidate USP30 deletion by protein levels; (7) examine the role of USP30 in KRT8+ intermediate\ncells and bronchiolar stem/progenitor cells.\nThese new or updated data are shown in Figs 2G, S1E, S1H, S1J, S1K, S2A-S2F, S3D-S3I,\nS4F-S4I, S5C, S5K, S5L, S6A-S6F, S8A-S8C, and S9. We have also revised the manuscript\ntext with necessary corrections. We believe that all concerns/questions raised by the reviewers\nhave been addressed, and the revised manuscript is significantly improved. Below are our\npoint-by-point responses to the reviewers' comments:\nReviewer #1 (Remarks to the Author)\nThis manuscript identifies USP30 as a novel therapeutic target in lung injury. The authors\nemploy both global and AT2-specific genetic knockout models, providing compelling evidence\nthat USP30 inhibition promotes lung regeneration in diverse injury contexts, including\nbleomycin-induced fibrosis and influenza-induced pneumonia. Mechanistically, USP30\ndepletion reduces AT2 cell apoptosis via VDAC1 following injury while enhancing their\nproliferative capacity, thereby limiting alveolar damage and promoting regeneration. Overall,\nthe study provides important insights into USP30 as a regulator of alveolar repair and highlights\nmitochondrial dysfunction as a promising therapeutic avenue, but several points require\nclarification and further analysis.\nMajor Points:\n1. Clarify whether USP30 deletion affects mesenchymal/fibroblast populations (apoptosis,\nproliferation, or differentiation) and whether fibroblasts show transcriptional changes in scRNA-\nseq.\nResponse: We thank the reviewer for the valuable suggestions. In response, we have\ninvestigated the impact of USP30 deletion on mesenchymal populations through both in vitro\nfunctional assays and in vivo transcriptome analysis. These data have been incorporated into\nthe revised manuscript as Figures S4F-S4I, S1J and S1K.\n1) In vitro functional assays: Primary lung fibroblasts were isolated from Usp30 KO and WT\nmice and treated with TGF-β1 for 48 hours to assess TGF-β1-induced myofibroblast\ndifferentiation (ACTA2+). To evaluate cell proliferation, the cells were cultured with or without\n10% FBS to measure EdU incorporation, and to examine apoptosis, the cells were cultured\nunder serum-free condition to measure Caspase-3/7 activity. Our results showed no\nsignificant differences between KO and WT fibroblasts in terms of myofibroblast\ndifferentiation, proliferation, or apoptosis (Figures A–C).\n2) In vivo scRNA-seq analysis: We analyzed lung fibroblast subpopulations using scRNA-\nseq dataset from Usp30 KO and WT mice 18 days post bleomycin injury. Usp30 deletion\ndid not significantly affect gene expression related to apoptosis, proliferation, or\ndifferentiation in any individual subpopulation (Figure D). However, Usp30 KO lungs\nexhibited a reduced proportion of \"Proliferating\" and \"Peribronchial\" fibroblast clusters,\nwhich were highly enriched for the pathogenic fibroblast marker Cthrc1, Postn and the\nmyofibroblast marker Acta2 (Figures E and F). This observation is consistent with the less\nsevere fibrosis phenotype in Usp30 KO mice.\nTogether, these results along with Figures S4C-S4E show that Usp30 deletion does not\nsignificantly impact lung fibroblast proliferation, activation, apoptosis or myofibroblast\ndifferentiation, indicating the reduced fibrotic burden probably is not due to cell-autonomous\neffects on fibroblasts.\nFigure legends: (A) Representative immunofluorescence images and quantification of ACTA2+ cells in\nprimary lung fibroblasts from WT and Usp30 KO mice cultured with or without TGF-β1 (4 ng/mL, 48 hr)\n(n=6 biological replicates / group). Scale bars: 100μm. (B) EdU incorporation assay (10 μM, 4 hr) in\nfibroblasts cultured under serum-free or 10% FBS conditions (n=8 biological replicates / group). Scale\nbars: 100μm. (C) Measurement of Caspase-3/7 activity in serum-free conditions (n=6 biological replicates\n/ group). (D) Violin plots of module scores for gene signatures across fibroblast subtypes. (E) Dot plot\ndisplaying expression of fibroblast markers across each distinct subcluster. (F) Proportion of fibroblast\nsubpopulations identified by scRNA-seq in bleomycin-injured lungs. Data in A-C represent mean ± SEM.\np values were analyzed by two-way ANOVA (A, B) and Student's t-test (C).\n2. Assess whether mitochondrial functions beyond mitophagy (e.g., oxidative phosphorylation)\nare altered in scRNA-seq data.\nResponse: We appreciate the reviewer’s constructive suggestions. In response, we have\nconducted a comprehensive scRNA-seq analysis of AT2 cells post bleomycin injury, specifically\nfocusing on mitochondrial functions. Usp30 deletion resulted in increased expression of\nPpargc1a, the master regulator of mitochondrial biogenesis as well as mitochondrial genes that\nencode for essential components of the mitochondrial respiratory chain (Figure A). Moreover,\nUsp30-KO AT2 cells exhibited higher mitochondrial OXPHOS levels, while glycolysis and\nmitochondrial stress markers were reduced compared to WT cells (Figure B). In contrast, WT\ncells displayed a \"retrograde response\"1 signature. characterized by upregulation of nuclear-\nencoded OXPHOS subunits, elevated mitochondrial stress markers (Hspd1, Hspa9), and\nreduced mitochondrial-encoded transcripts. These transcriptional changes were further\nsupported by increased oxygen consumption rates and ATP production as examined by the\nseahorse assay (Fig. 2A and 2B).\nTo further investigate whether Usp30 KO in AT2 cells regulates mitochondrial function, we\nperformed bulk RNA-seq of lineage-traced WT and Usp30-KO AT2 cells flow-sorted from\nuninjured WTΔSftpc and Usp30ΔSftpc mice. Gene Set Enrichment Analysis (GSEA) revealed\nenrichment of mitochondrial translation, respirasome assembly and proton motive force-driven\nmitochondrial ATP synthesis in Usp30-KO AT2 cells (Figure C). Together, these findings\ndemonstrated Usp30 KO improves mitochondria function both at baseline conditions and post\nbleomycin injury. These data have been incorporated into the revised manuscript as Figures\nS2E, S2F and S6A.\nFigure legends: (A) Dot plot of representative genes corresponding to mitochondrial biogenesis, dynamics\nand metabolic modules from scRNA-seq. (B) Violin plots of module scores for glycolysis, mitochondrial\nstress, nuclear-encoded OXPHOS, and mitochondrial-encoded OXPHOS signatures in AT2 cell scRNA-\nseq dataset. (C) GSEA of lineage-traced AT2 cells from WTΔSftpc and Usp30ΔSftpc mice under homeostatic\nconditions.\n3. Determine if USP30 is expressed in transient cell states (DAPT/PATS/K8+) and whether\nknockout in AT2 cells impacts these populations post-injury.\nResponse: We thank the reviewer for the valuable suggestions. In response, we have analyzed\nUsp30 expression in transient cell states and quantified KRT8+ cells in both global and AT2-\nspecific Usp30 KO mice (Figures S1H, S3D, S5K and S5L in the revised manuscript).\nUsp30 is universally expressed in all the cell types of mouse lungs, including in KRT8+\nintermediate cells (DAPT/PATS/ADI) (Figure A). We observed a significant increase in KRT8+\ncells in the alveolar regions of Usp30 KO lungs (Figure B), along with a higher percentage of\nSFTPC-lineage-traced KRT8+ cells and RAGE+ AT1 cells in AT2-specific Usp30 KO lungs\n(Figures C and D). These findings suggest that Usp30 KO promotes AT2 cell proliferation and\nfacilitates their differentiation of AT2 into mature AT1 cells, thereby enhancing alveolar\nregeneration.\nFigure legends: (A) Dotplot of Usp30 expression in all cell types from scRNA-seq. (B) Representative\nimmunofluorescence images and quantification of KRT8+ cells in lung sections from WT and Usp30-/-\nmice on day 18 post-bleomycin (WT: n=9 mice; KO: n=8 mice). Scale bars: 100 μm. (C) Representative\nimmunofluorescence images and quantification of Sftpc-lineage-traced cells (tdTomato+) expressing\nKRT8 in Usp30ΔSftpc and control mice on day 14 (n=7 mice / group). Scale bars: 100 μm. (D)\nRepresentative immunofluorescence images and quantification of lineage-labeled cells expressing RAGE\non day 14 (n=7 mice / group). Data in B-D represent mean ± SEM. p values were analyzed by Student's\nt-test.\n4. Evaluate potential effects of USP30 deletion on distal bronchiolar stem/progenitor cells\n(secretory cells, BASCs, LNEPs).\nResponse: We thank the reviewer for the valuable suggestions. In response, we have\nperformed the following experiments to evaluate the potential role of USP30 in these cell types:\n1) Secretory cells: Since there are no suitable surface markers to enrich secretory cells from\nUSP30 KO mice, we used Scgb1a1-CreER; R26-mTmG mice to flow sort secretory cells,\nwhich were then cultured in 3D organoids and treated with the USP30 inhibitor MF094.\nMF094 treatment did not significantly affect colony formation or cell proliferation (Figures\nA–C).\n2) BASCs: Bronchioalveolar Stem Cells (BASCs) are rare at the bronchioalveolar duct\njunction, making it difficult to isolate and culture them. Instead, we examined BASC cell\n(ZsGreen+ tdTomato+) number in the Scgb1a1-CreER; Sftpc-DreERT2; H11-ZSGT mice\non day 8 post bleomycin, which remained comparable between MF094-treated and vehicle\ngroups (Figure D).\n3) LNEPs: LNEPs give rise to KRT5+ cells in the damaged alveoli following influenza\ninfection.2, 3 These cells are also rare, making it difficult to isolate and culture them. As an\nalternative, we assessed KRT5+ cell expansion in WT and USP30 KO mice to indicate\nLNEP activity. No differences were observed between WT and KO groups in KRT5+ cell\nexpansion post influenza (Figure E)\nIn summary, due to the limited experimental tools and systems available, we were unable to\nfully evaluate the role of USP30 in the bronchiolar stem/progenitor cells. Our preliminary results\nsuggest that USP30 deletion or inhibition seems not significantly affect the proliferation of\nsecretory cells, BASCs or LNEPs. However, further studies are needed to confirm these\nfindings. We would be happy to include these data as a supplementary material in the\nmanuscript if the reviewer feels that it would enhance the manuscript.\nFigure legends: (A) Schematic diagram of the experimental design for secretory cell organoid culture. (B\nand C) Quantification of Colony Formation Efficiency (CFE) (Scale bars: 500 μm) (B) and EdU\nincorporation (Scale bars: 20 μm) (C) in Scgb1a1-trace organoids treated with Vehicle or MF094 (n=4\ndroplets / group). (D) Quantification of BASCs (ZsGreen+ tdTomato+) in Scgb1a1-CreER; Sftpc-DreERT2;\nH11-ZSGT mice (n=3 mice / group) on day 8 post bleomycin (Scale bars: 50 μm). White arrows indicate\nBASCs. (E) Representative immunofluorescence images and quantification of KRT5+ cells in WT and\nUsp30-/- lungs on day 14 post-influenza (n=12 mice / group). Scale bars: 200 μm. Data in B-E represent\nmean ± SEM. p values were analyzed by Student's t-test.\n5. Provide clarification on the design of the floxed allele and possible reasons for limited\nrecombination efficiency in AT2-specific KO mice (Figure S3B). Could this be related to the\nsequence length between two loxP sites in the floxed USP30 allele, making excision less\nefficient even with the robust Sftpc-CreERT2 driver?\nResponse: We thank the reviewer for the insightful suggestion. We have sequenced the\ngenome of AT2-specific KO mice and found the sequence length between two loxP sites is\n1,276 bp (Figure A). According to literature, optimal recombination is achieved when loxP sites\nare spaced by 1.0–4.0 kb.4, 5\nWe then tried to detect USP30 protein levels to determine the knockout efficiency. However,\ndue to the low abundance of endogenous USP30 levels, we could not detect USP30 by western\nblotting. We employed Tyramide Signal Amplification (TSA) in immunofluorescence staining to\namplify the signals. USP30 has been reported to primarily localize to mitochondria and\nperoxisomes.6 TSA staining showed mostly cytoplasm signals along with cell surface signals.\nQuantification of the cytoplasm signals confirmed ~50% decrease of protein levels in lineage-\ntraced AT2 cells (Figure B).\nFigure legends: (A) Genomic sequencing analysis validating the 1,276 bp distance between loxP sites in\nAT2-specific KO mice. (B) Representative immunofluorescence images and quantification of USP30\nlevels specifically within uninjured Sftpc-traced AT2 cells (tdTomato+) (n = 8 fields pooled from 3 mice\n/ group). Scale bars: 10 μm. Data in B represent mean ± SEM. p values were analyzed by Student's\nt-test.\n6. Include lineage-tracing data (tdTomato) in AT2-specific KO experiments (Figure3) to better\ndistinguish AT2-derived regeneration from bronchiolar stem cell contributions. More precise\nquantification of AT2 proliferation (tdT+ vs tdT–) is needed.\nResponse: We thank the reviewer for the suggestions. We have now quantified EdU\nincorporation in tdT- AT2 cells. While Usp30-KO tdT+ AT2 cells exhibited significantly higher\nproliferation rates compared to WT tdT+ AT2s (Fig. 3M), tdT- AT2 cells in Usp30ΔSftpc mice\nshowed comparable proliferation rates compared with that in WT mice (Figure A). To evaluate\nthe relative contribution of surviving AT2 and bronchiolar stem/progenitor populations in the\nproliferating AT2 cells post influenza infection, we analyzed the lineage composition of the\nproliferating AT2 pool. tdT+ AT2 cells constituted >80% of total EdU+ AT2 cells in both WT and\nUsp30 cKO mice (Figure B). We would be happy to include these data as a supplementary\nmaterial in the manuscript if the reviewer feels that it would enhance the manuscript.\nFigure legends: (A) Quantification of EdU incorporation specifically within tdT- AT2 population in Sftpc-\nCreERT2 mice (PBS: n=3 mice / group; influenza: n=13 mice / group). (B) Stacked bar graph showing the\nproportion of lineage-labeled (tdT+) and non-labeled (tdT-) cells within the total EdU-positive AT2\npopulation. Data represent mean ± SEM. p values were analyzed by two-way ANOVA (A).\nMinor Points:\n1. The statement that USP30 KO “does not affect” fibroblasts or macrophages may be too\nstrong according to limited data here; consider rephrasing.\nResponse: We appreciate the reviewer’s suggestion and we have revised the text accordingly\nin Line 252–260 and Line 437.\nReviewer #2 (Remarks to the Author):\nWu et al. investigate the role of USP30-mediated mitophagy in promoting alveolar regeneration\nfollowing lung injury. The authors used genetic and pharmacological approaches targeting\nUsp30 to demonstrate that loss of USP30 enhances mitophagy and restores mitochondrial\nfunction in AT2 cells, thereby protecting them from apoptosis and promoting regenerative\ncapacity. The findings are interesting and provide a fresh insight into the cellular mechanisms\nof tissue repairing. The study nicely shows that Usp30 deletion reduces AT2 apoptosis and\npromotes proliferation across multiple lung injury models. It can be further strengthened to\nexamine the link between USP30 loss and changes in the AT2 cell transcriptomic profile\nwith or without injury, providing evidence how USP30 loss enhances AT2 stem cell\nactivity at the molecular level. A few other minor issues are listed below.\nResponse: We appreciate the reviewer’s valuable suggestions. To understand the molecular\nmechanism underlying enhanced stem cell activity, we have performed bulk RNA-seq on\nlineage-traced AT2 cells isolated from Usp30ΔSftpc mice and controls, both at baseline and on\nday 14 post bleomycin and influenza injury. These data have been incorporated into the revised\nmanuscript as Figures S6 and S8.\nAt baseline, Usp30-deficient AT2 cells showed enrichment of proton motive force-driven\nmitochondrial ATP synthesis, mitochondrial translation and respirasome assembly (Figures A\nand B), in support of improved mitochondria function. Pathway analysis in Usp30-KO AT2 cells\nat baseline showed enrichment of Notch, Wnt, MAPK, and PI3K-AKT signaling, which are\nimportant for stem cell activity and cell proliferation (Figure C).\nFollowing bleomycin injury, Usp30-KO AT2 cells downregulated immune responses and\nsenescence markers (Cdkn1a, Cdkn2a, Cdkn2b) while upregulating AT2 identity markers (Sftpc,\nEtv5) (Figures D-F). Notably, multiple genes associated with Wnt/β-catenin signaling, FGF\nsignaling, and Hippo-YAP signaling, which are critical for AT2 stem cell activity,7-11 were\nupregulated, including Axin2, Fzd1/2, Ctnnb1, Fgfr2, and Yap1. Pathway analysis showed\nenrichment of PI3K-AKT and MAPK signaling.\nFollowing influenza infection, Usp30-KO AT2 cells exhibited a marked upregulation of cell cycle\nand mitotic pathways, along with a downregulation of immune response-related genes, such\nas AT2-intrinsic chemokines and alarmins (Cxcl2, Ccl3, S100a8, Il1b), consistent with the\nattenuated lymphocyte activation and recruitment observed in GSEA (Figures G-I). Pathway\nanalysis showed enrichment of PI3K-AKT and MAPK signaling.\nThese findings suggest Usp30 KO in AT2 cells improved mitochondrial function and\nupregulated multiple signaling pathways, leading to enhanced stem cell activity.\nFigure legends: (A-C) Transcriptomic analysis of lineage-traced AT2 cells from WTΔSftpc and Usp30ΔSftpc\nmice under homeostatic conditions. Shown are the dotplot of GSEA (A), cumulative distribution function\n(CDF) analysis of mitochondrial gene expression (B), and barplot of selected signaling pathways from\nKEGG analysis (C) (|log FC| ≥ 0.58, padj < 0.05) (n=3 biological replicates / group). (D-F) Transcriptomic\n2\nanalysis of lineage-traced AT2 cells 14 days post-bleomycin injury. Shown are the GSEA (D), heatmap\ndisplaying the relative expression (row-scaled Z-score of log TPM) of selected genes in sorted AT2 cells\n2\n(E), and barplot of selected signaling pathways from KEGG analysis (F) (|log FC| ≥ 0.58, padj < 0.05)\n2\n(n=5 biological replicates / group). (G-I) Transcriptomic analysis of lineage-traced AT2 cells 14 days post-\ninfluenza infection. Shown are the GSEA (G), heatmap displaying the relative expression (row-scaled Z-\nscore of log TPM) of selected genes in sorted AT2 cells (H), and barplot of selected signaling pathways\n2\nfrom KEGG analysis (I) (|log FC| ≥ 0.58, padj < 0.05) (n=4 biological replicates / group).\n2\n1. The authors performed scRNA-seq for Usp30-/- and control mice followed by bleomycin\nchallenge. It is interesting that no pathological fibroblasts were identified. This reviewer wonder\nif the authors examine pathological fibroblasts markers Cthrc1 and Postn?\nResponse: We thank the reviewer for the helpful suggestions. Cthrc1 expression was enriched\nin the \"Proliferating fibroblast\" cluster, while Postn was enriched together with Acta2 in both\n\"Peribronchial fibroblast\" and \"Proliferating fibroblast\" clusters (Figure A), confirming that\n\"Peribronchial fibroblast\" and \"Proliferating fibroblast\" clusters resemble Cthrc1+ and Postn+\npathological fibroblasts. Usp30 KO mice exhibited a marked reduction in the proportion of\n\"Peribronchial fibroblast\" and \"Proliferating fibroblast\" clusters compared to WT (Figure B).\nThese data have been included in the revised manuscript as Figures S1J and S1K.\nFigure legends: (A) Dot plot displaying expression of fibroblasts marker across each distinct subcluster.\n(B) Proportion of fibroblast subpopulations identified by scRNA-seq in bleomycin-injured lungs.\n2. Figures S1I and S1J: Saline-treated controls can be further analyzed. Data on mitochondrial\nnumber and function in uninjured lungs would strengthen the baseline comparison.\nResponse: We appreciate the reviewer’s valuable suggestions. In response, we have examined\nmitochondrial number and architecture in saline-treated controls using scanning electron\nmicroscopy (SEM). Mitochondrial number in AT2 cells were comparable between WT and\nUsp30-KO groups both at baseline and after bleomycin injury (Figure A). Usp30-/- mitochondria\ndisplayed a smaller, more condensed cross-sectional area compared to WT controls under both\nat baseline conditions and following injury (Figure A and B). These results have been included\nin the revised manuscript as Fig. S2A-S2B.\nFigure legends: (A) Representative SEM images and quantification of mitochondrial cross-sectional area\nand count in AT2 cells of saline and bleomycin-treated lungs (n ≥ 100 representative fields of view pooling\nfrom 3 mice / group). Scale bars: 1 μm (overview); 300 nm (magnified view). (B) 3D reconstruction of\nmitochondrial networks from serial SEM sections. Scale bars: 2μm. Data in A represent mean ± SEM. p\nvalues were analyzed by two-way ANOVA (A).\n3. Figures 3A–3C: It is unclear whether observed mitochondrial morphology and gene\nexpression changes are specifically from AT2 cells. Additional validation such as AT2-specific\nisolation or imaging would clarify this point.\nResponse: We apologize for the confusion. Figures 3A-3C are specifically from AT2 cells. AT2\ncells were identified in the EM images based on their characteristic lamellar bodies (LBs) (Fig.\n3B). RNA-seq was performed on flow-sorted AT2 cells (Fig. 3C). We have revised the\nmanuscript text for clarity (Line 310- Line 316).\n4. Figure 3C heatmap and KEGG analysis: The comparison focuses on time points rather than\ngenotypes. It would be more informative to directly compare WT and Usp30⁻/⁻ gene expression\nprofiles to identify pathways altered by USP30 loss.\nResponse: We thank the reviewer for the constructive suggestions. In response, we have\nperformed bulk RNA-seq on lineage-traced WT and Usp30-KO AT2 cells isolated from\ninfluenza-infected WTΔSftpc and Usp30ΔSftpc mice on day 14 to directly compare their\ntranscriptional profiles.\nUsp30-KO AT2 cells exhibited a marked upregulation of cell cycle and mitotic pathways, along\nwith a downregulation of immune response-related genes (Figures A-C). The suppressed\ninflammatory profile was characterized by the reduction of AT2-intrinsic chemokines and\nalarmins (Cxcl2, Ccl3, S100a8, Il1b), consistent with the attenuated lymphocyte activation and\nrecruitment pathways observed in GSEA. These results indicate that Usp30 deletion promotes\nAT2 cell proliferation, contributing to the resolution of inflammation. These data have been\nincorporated into the revised manuscript as Figures S8 in revised manuscript.\nFigure legends: (A-C) Transcriptomic analysis of lineage-traced AT2 cells 14 days post-influenza infection.\nShown are the GSEA (A), heatmap displaying the relative expression (row-scaled Z-score of log TPM) of\n2\nselected genes in sorted AT2 cells (B), and barplot of selected signaling pathways from KEGG analysis\n(C) (|log FC| ≥ 0.58, padj < 0.05) (n=4 biological replicates / group).\n2\n5. The authors cultured AT2 organoids in AMM-based medium as described in Ref 91. It\nseems that AT1 cell differentiation rarely happens if cultured in AMM. It is interesting that the\nauthors detected quite a number of AT1 cells in this culture system (Fig. 2L, M). Did the\nauthors modify the medium?\nResponse: We thank the reviewer for raising this question. Yes, we modified the medium to\ninduce AT1 differentiation by withdrawing CHIR99021 and IL-1β from the AMM medium. The\ndetailed protocol was described in the methods sections.\nReviewer #3 (Remarks to the Author):\nStudies by Wu et al, analyzes the susceptibility to lung injury and fibrosis by inhibition of USP30\nusing genetic and pharmacological approaches. Studies analyze the bleomycin induced lung fibrosis\nand the influenza pneumonia murine models. Studies show that inhibition of USP30 in AT2 cells\nis sufficient to increase resilience against lung injury. Investigators hypothesize inhibition of\nUSP30 promotes AT2 proliferation by an ROS mediated mechanisms and protection against\napoptosis by reducing VDAC protein levels. The studies are relevant to the field and have\ntranslational potential. However, there are some concerns that need to be addressed.\n1. The significance of the studies, especially the translational potential, will be enhanced with\nevidence of higher expression of USP30 in AT2 cells of human lung fibrotic diseases like IPF.\nSimilarly, if changes in ubiquitination and protein levels of VDAC1 observed in the current\nmouse studies are relevant for the human fibrotic disease.\nResponse: We appreciate the reviewers’ valuable suggestions. In response, we have\nperformed western blot of USP30 and VDAC1 in human IPF and healthy donor lungs. Our\nresults showed significantly elevated protein levels of USP30 and its substrate, VDAC1, in IPF\nlungs compared to healthy donors (Figure A). Due to the lack of suitable antibodies for human\nUSP30 and VDAC1 for immunofluorescence staining, we analyzed a publicly available scRNA-\nseq dataset of IPF lungs (GSE136831)12 to examine USP30 and VDAC1 expression. USP30\nmRNA levels were upregulated in multiple cell types, including AT2 cells, in IPF lungs, whereas\nVDAC1 mRNA levels were not increased in AT2 cells from IPF lungs (Fig. B and C), This\nsuggest that the regulation of VDAC1 by USP30 occurs at the post-translational level rather\nthan through transcriptional regulation. The western blot results have been incorporated into\nthe revised manuscript as Fig. 2G. We would be happy to include the transcriptome data as a\nsupplementary material in the manuscript if the reviewer feels that it would enhance the\nmanuscript.\nFigure legends: (A) Representative immunoblots and quantification of USP30, VDAC1, and fibrotic\nmarkers (ACTA2, COL1A1) in whole lung lysates from healthy donors (Normal: n=4) and IPF patients\n(n=6). β-Actin served as the loading control. (B and C) Dot plots visualizing USP30 (B) and VDAC1 (C)\nmRNA expression across distinct epithelial cell subsets derived from the GSE136831 dataset. Data in A\nrepresent mean ± SEM. p values were analyzed by Student's t-test.\n2. scRNAseq data do not align with the other set of data of the manuscript. For example, the\nproportion of myofibroblasts that the authors show increase by IFA are not shown, nor the\ncluster of fibrotic fibroblasts CTHRC1+ that produce high levels of ECM. These changes will\nconfirm the effect in fibrosis by the absence of USP30. Similarly, it would be important to define\nthe cluster of proliferating AT2 to confirm the EdU and organoid experiments. Additionally, there\nis lack of AT1 cell cluster. The AT2-AT1 cluster shown in the UMAP data has a limited\nproportion in comparison with the abundant Ager+ staining shown in Figure 2.\nResponse: We thank the reviewer for raising these points. In response, we have analyzed the\nscRNA-seq data to validate our experimental findings.\n1) Fibroblasts: we analyzed lung fibroblast subpopulations in the scRNA-seq dataset.\nPathologic fibroblast marker Cthrc1 was enriched in the \"Proliferating fibroblast\" cluster,\nwhile myofibroblast marker Acta2 was enriched in both \"Peribronchial fibroblast\" and\n\"Proliferating fibroblast\" clusters (Figure A). Usp30 KO mice exhibited a marked reduction\nin the proportion of \"Proliferating fibroblast\" and \"Peribronchial fibroblast\" clusters\ncompared to WT (Figure B). Usp30 deletion did not significantly affect gene expression\nrelated to apoptosis, proliferation, or differentiation in any individual fibroblast\nsubpopulation (Figure C), which is consistent with our in vitro functional assays. These\nfindings are included in the revised manuscript as Fig. S1J, S1K and S4I.\nFigure legends: (A) Dot plot displaying expression of fibroblast markers across each distinct subcluster.\n(B) Proportion of fibroblast subpopulations identified by scRNA-seq in bleomycin-injured lungs. (C) Violin\nplots of module scores for gene signatures across fibroblast subtypes.\n2) AT2 Proliferation: We analyzed AT2 cells in the scRNA-seq dataset and found the\nproportion of Mki67+ AT2 cells was decreased in Usp30-KO AT2 cells compared to WT\ncontrols (Figures D and E). This discrepancy likely reflects different timepoints of the\nexperiments. The scRNA-seq was performed on day 18 post injury, which is fibrotic phase\nin bleomycin model, while EdU assay was performed on day 8 when endogenous AT2\ncells are highly proliferating (epithelial repair phase). While Usp30-KO AT2 cells exhibited\nhigher proliferation rates during repair phase, the lower proportion of proliferating AT2 cells\nin KO mice on day 18 suggests a faster return to homeostatic quiescence.\nFigure legends: (D) Feature plots showing the expression of the proliferation marker Mki67 in AT2 cells\nfrom WT and Usp30 KO lungs on day 18 post-bleomycin injury. (E) Quantification of the percentage of\nMki67+ cells within AT2 cell population.\n3) Lack of AT1 Cells: AT1 cells are thin, large and fragile, which make them particularly\nsusceptible to the tissue dissociation methods required for single-cell suspensions.13\nHowever, immunofluorescence staining was effective in capturing these cells. In our\nscRNA-seq, we did not identify a distinct population solely expressing the mature AT1\nmarkers. Instead, we observed a population expressing both AT1 marker Ager and the\nintermediate marker Krt8, which we defined as “AT2-to-AT1 cells” (Figure F).\nFigure legends: (F) UMAP plot of epithelial cell subpopulations (left) and feature plots showing the\nexpression of AT2 (Sftpc), transition (Krt8), and AT1 (Ager, Hopx, Aqp5, Pdpn) markers (right) in\nbleomycin-injured lungs on day 18.\n3. Interestingly, studies in Figure 2 and 2S show that AT2 cells of WT mice in naïve conditions\nhave active apoptosis in contrast to AT2 from USP30 KO. Authors suggest that this apoptosis\nis related to high levels of VDAC protein. It is unclear what is the stimuli for overexpression of\nVDAC1 in naïve healthy AT2 cells to mediate apoptosis. Furthermore, transcriptomic data is\nneeded to demonstrate that changes in VDAC in USP30 deficient AT2 cells are not associated\nwith transcriptional changes and only are related to changes in ubiquitination and protein\ndegradation.\nResponse: We thank the reviewer for the helpful suggestions. In Fig. 2C, we measured the\napoptosis of primary AT2 cells isolated from WT and Usp30-KO mice and treated with or without\nbleomycin in culture. In naïve conditions, apoptosis levels reflect the inherent stress of culturing\nprimary cells, with less than 3% cells undergoing apoptosis. In contrast, bleomycin treatment\ninduced ~15% cells undergoing apoptosis. Usp30 KO significantly inhibited apoptosis both\nunder baseline and post bleomycin injury.\nVDAC1 is a key player of mitochondrial-dependent apoptosis by forming channels on the outer\nmitochondrial membrane, facilitating the release of cytochrome c into the cytosol.14, 15\nOverexpression of VDAC1 in HEK293 T cells resulted in less than 0.5% apoptotic cells under\nbaseline conditions, whereas approximately 3% of cells underwent apoptosis following\nbleomycin injury (Fig. S3C in the revised manuscript). We think the observed apoptosis in naïve\nconditions is primarily due to the stress associated with 2D culture.\nTo investigate the regulation of VDAC1 by USP30, we performed transcriptional analysis. No\nsignificant differences were observed in Vdac1 mRNA levels between WT and KO cells under\nbaseline conditions, either in the bulk cell RNA-seq of FACS-sorted primary AT2 cells (Figure\nA) or primary AT2 cells in 2D culture (Figure B). These results together with the western blots\nin bleomycin and IPF lungs (Fig. 2F and 2G in the revised manuscript) suggest that the\nregulation of VDAC1 by USP30 occurs at the post-translational level. We would be happy to\ninclude these data as a supplementary material if the reviewer feels that it would enhance the\nmanuscript.\nFigure legends: (A) Vdac1 TPM values from bulk RNA-seq of FACS-sorted AT2 cells (n=3 biological\nreplicates / group). (B) qRT-PCR analysis of Vdac1 and Usp30 in primary AT2 cells cultured in 2D\nmonolayers (n=5 biological replicates / group). Data represent mean ± SEM. p values were analyzed by\nStudent's t-test.\n4. The high levels of mtROS in AT2 from USP30 KO mouse are in contrast to the increase in\nmaximal respiration shown in the seahorse studies for KO cells. High maximal OCR generally\nis associated with a state of low mtROS generation and mitochondrial fitness. Furthermore, it\nis unclear why there are not changes in the mitochondrial TMRM and mitochondrial mass\nbetween mouse strains if mitophagy is induced differently between groups. Simultaneous\nanalysis of these two parameters might help to give a better understanding as well as\ncomplementary markers of autophagy like p62 protein levels, and ubiquitination of Parkin to\nanalyze mitophagy. Furthermore, studies of mitophagy flux in the AT2 cells might be also useful.\nResponse: We appreciate the reviewer’s constructive suggestions. Since OCR was measured\nin cultured primary AT2 cells, we have further examined the dynamic changes of ROS levels,\nmitochondrial TMRM, mitochondrial mass and mitophagy flux in these cells. These data have\nbeen incorporated into the revised manuscript as Figures S3E-S3I.\n1) ROS levels: Usp30-KO cells exhibited moderately elevated baseline mtROS and cytosolic\nROS levels, along with a transient increase of mtROS following bleomycin injury, which\nthen decreased (Figures A and B). In contrast, WT cells showed an accumulation of\ncytosolic ROS after injury. These findings suggest Usp30-KO cells effectively mitigate\ninjury-induced oxidative stress.\n2) TMRM and Mitochondrial Mass: No significant differences in TMRM and mitochondria\nmass were observed between WT and Usp30-KO AT2 cells at baseline (Figures C and D).\nHowever, following injury, mitochondria mass gradually increased until 6 hours and then\ndeclined at 8 hours in WT cells, while KO cells exhibited an accelerated decline starting at\n4 hours, suggesting enhanced mitophagy. Consistent with this, KO cells had increased\nTMRM at 6 hours post injury.\n3) Mitophagy Flux: Based on the dynamic changes observed in Figures A-D, we performed\nwestern blotting for p62 and LC3B to examine the mitophagy flux at 4 hours post injury.\nBafilomycin A1 (BafA1) inhibits lysosomal acidification, resulting in a blockage in\nautophagic flux and accumulation of autophagosomes. In the absence of BafA1, Usp30 KO\ncells exhibited lower levels of p62 and higher levels of LC3B-II compared to WT cells\n(Figure E), suggesting enhanced mitophagy. In the presence of BafA1, Usp30 KO cells\naccumulated significantly higher levels of LC3B-II and p62, indicating a higher rate of\nautophagosome formation and fusion with lysosomes.\nIn summary, Usp30-KO cells exhibited enhanced mitophagy, increased mitochondrial\nbiosynthesis and elevated ATP synthesis. After injury, they displayed a transient increase in\nmtROS followed by a decline. This phenomenon is consistent with the concept of mitohormesis,\nwhere mild mitochondrial stress, such as a brief increase in mtROS, induces beneficial adaptive\nresponses that enhance cellular function and cause a long-term reduction of oxidative stress.16-\nbleomycin (2 mg/mL) for 0h–8h. Panels show mt-ROShigh population (A), cROS MFI (B), mitotracker MFI\n(C), TMRMhigh population (D) (n=8 biological replicates / group). (E) Representative immunoblots of\nautophagic flux markers (LC3B, p62) in primary AT2 cells. Cells were treated with bleomycin (2 mg/mL)\nfor 4 hours, with or without Bafilomycin A1 (BafA1, 100 nM) for the final 2 hours. β-Actin served as the\nloading control. Data in A-D represent mean ± SEM. p values were analyzed by two-way ANOVA (A-D).\n5. Figure 1M do not show markers of cell type to define if AT2 cells have been analyzed for the\ncolocalization of mitochondria and LC3B. it seems that USP30 deficiency increase levels of\nLC3B.\nResponse: We thank the reviewer for the suggestion. In response, we have included AT2\nspecific marker SFTPC in the immunofluorescence staining by optimizing a four-color\nimmunofluorescence protocol (Mito-Dendra2, SFTPC-Cy5, LC3B-Cy7 and DAPI). These data\nhave been incorporated into the revised manuscript as Figures S2C and S2D.\n1) AT2-Specific Mitophagy Analysis: We quantified the mitophagy index (ratio of LC3B+\nmitochondrial area to total mitochondrial area) specifically within SFTPC+ AT2 cells.\nUsp30-KO AT2 cells exhibited a significantly higher mitophagy index compared to WT\ncontrols under both homeostatic and injury conditions (Figures A and B).\n2) LC3B Signal Intensity: We quantified LC3B intensity in AT2 cells and confirmed that\nsignal intensity was elevated in the Usp30 KO group (Figure C). This increase reflects\nenhanced recruitment of the autophagy machinery driven by the stabilization of ubiquitin\nchains on damaged mitochondria, suggesting enhanced autophagic flux rather than a\nblockage. As further validated by LC3B western blotting (Figure S3I in the revised\nmanuscript), lysosomal inhibition induced a significant additional accumulation of LC3B-II\nin KO cells.\nFigure legends: (A and B) Representative multicolor immunofluorescence images (A) (Scale bars: 5μm)\nand quantification (B) of mitophagy index in lung sections from Usp30 KO and WT mice expressing the\nmito-Dendra2 reporter (n ≥ 70 representative fields pooled from 3 mice / group). Sections were co-stained\nfor SFTPC (Cy5) and LC3B (Cy7). Mitophagy index was calculated as the ratio of LC3B+ mitochondrial\narea to the total mitochondrial area specifically within SFTPC+ AT2 cells. (C) Quantification of the mean\nfluorescence intensity (MFI) of LC3B signal specifically within the SFTPC+ AT2 cell population. Data\nrepresent mean ± SEM. p values were analyzed by two-way ANOVA.\n6. The authors in line 162 suggest enhanced mitochondrial biosynthesis in AT2 from USP30\nKO suggested by mitochondrial morphology and number. Complementary studies of markers\nof mitochondrial biogenesis and dynamics are recommended to support the statement.\nResponse: We thank the reviewer for the valuable suggestion. In response, we have examined\ngene expression related with mitochondrial biogenesis and dynamics using scRNA-seq data.\nUsp30-KO AT2 cells displayed increased expression of Ppargc1a, the master regulator of\nmitochondrial biogenesis, and replication/transcription machinery (Polrmt, Twnk) as well as\nhigher expression of mitochondrial genes that encode essential components of the\nmitochondrial respiratory chain (Figure A). GSEA analysis confirmed that enrichment of\nmitochondrial translation, respirasome assembly and proton motive force-driven mitochondrial\nATP synthesis in Usp30-KO AT2 cells (Figure B). Usp30-KO AT2 cells also exhibited\ndownregulation of fission genes (Fis1, Dnm1l) and fusion markers (Mfn2, Opa1), with the\nexception of Mfn1, which was upregulated. These data have been incorporated into the revised\nmanuscript as Figures S2E.\nFigure legends: (A) Dot plot of representative genes corresponding to mitochondrial biogenesis, dynamics\nand metabolic modules from scRNA-seq. (B) GSEA of lineage-traced AT2 cells from WTΔSftpc and\nUsp30ΔSftpc mice under homeostatic conditions.\n7. Figure 4B shows inhibition rate of the Compound MF094, however it will be more specific to\nshow the effect of the compound on the ubiquitination of a known mitochondrial protein target\nof USP30.\nResponse: We thank the reviewer for the helpful suggestion. In response, we have examined\nthe ubiquitination of phospho-Ser65-Ubiquitin (p-S65-Ub), which serves as a robust proxy for\nthe ubiquitination status of mitochondrial USP30 substrates.19 MF094 treatment resulted in a\ndose-dependent accumulation of p-S65-Ub in mouse AT2 cell line (MLE12 cells) treated with\nmitochondrial uncoupler Carbonyl cyanide m-chlorophenyl hydrazone (CCCP). This finding\nconfirms that MF094 effectively inhibits USP30 deubiquitinase activity by preserving ubiquitin\nchains on mitochondria, thereby enhancing the p-S65-Ub signal (Fig. S9 in the revised\nmanuscript).\nFigure legends: Representative immunoblots of Phospho-Ser65-Ubiquitin in MLE12 cells treated with\nvarious concentrations of MF094 and CCCP (10 μM). Tom20 and β-Actin served as loading controls.\nMinor concerns\n1. The measurements for OH-Pro for bleomycin and influenza pneumonia lung models have\nmore data points than other experimental variables, suggesting than more than 1 sample per\nlung mouse was analyzed. There is not a description in this respect on methods. Please clarify.\nResponse: We apologize for causing confusion. Each data point represents an independent\nbiological replicate, corresponding to a distinct mouse in the animal study. For the OH-Pro\nmeasurements shown in Fig. 1J and S5F, we pooled mice from 2 independent experiments, as\nthe OH-Pro window in bleomycin model typically shows less than a 2-fold difference.\n2. The USP30 protein expression and levels after deletion were not analyzed\nResponse: We appreciate the reviewer’s suggestion. We tried to detect USP30 protein levels\nin the mouse lungs. However, due to the low abundance of endogenous USP30 levels, we\ncould not detect USP30 by western blotting. We employed Tyramide Signal Amplification (TSA)\nin immunofluorescence staining to amplify the signals. USP30 has been reported to primarily\nlocalize to mitochondria and peroxisomes.6 TSA staining showed mostly cytoplasm signals\nalong with cell surface signals. Quantification of the cytoplasm signals confirmed significant\nprotein deletion in Usp30-/- lungs (Figure A) and specifically within lineage-traced AT2 cells in\ncKO mice (Figure B). These data have been incorporated into the revised manuscript as\nFigures S1E and S5C.\nFigure legends: (A) Representative immunofluorescence images (left) and quantification (right) of USP30\nsignal intensity in lung sections from uninjured WT and Usp30 KO mice (n = 10 fields pooled from 3 mice\n/ group). Scale bars: 10μm (B) Representative immunofluorescence images (left) and quantification (right)\nof USP30 levels specifically within uninjured Sftpc-traced AT2 cells (tdTomato+) (n = 8 fields pooled from\n3 mice / group). Scale bars: 10μm Data represent mean ± SEM. p values were analyzed by Student's t-\ntest.\nReference\n1. Butow, R.A. & Avadhani, N.G. Mitochondrial signaling: the retrograde response. Mol Cell 14, 1-\n15 (2004).\n2. Vaughan, A.E. et al. Lineage-negative progenitors mobilize to regenerate lung epithelium after\nmajor injury. Nature 517, 621-625 (2015).\n3. Xi, Y. et al. Local lung hypoxia determines epithelial fate decisions during alveolar regeneration.\nNat Cell Biol 19, 904-914 (2017).\n4. Erhardt, V. et al. Systematic optimization and prediction of cre recombinase for precise genome\nediting in mice. Genome Biol 26, 85 (2025).\n5. Bedolla, A.M. et al. A comparative evaluation of the strengths and potential caveats of the\nmicroglial inducible CreER mouse models. Cell Rep 43, 113660 (2024).\n6. Marcassa, E. et al. Dual role of USP30 in controlling basal pexophagy and mitophagy. EMBO\nRep 19 (2018).\n7. Nabhan, A.N., Brownfield, D.G., Harbury, P.B., Krasnow, M.A. & Desai, T.J. Single-cell Wnt\nsignaling niches maintain stemness of alveolar type 2 cells. Science 359, 1118-1123 (2018).\n8. Nabhan, A.N. et al. Targeted alveolar regeneration with Frizzled-specific agonists. Cell 186,\n2995-3012 e2915 (2023).\n9. Brownfield, D.G. et al. Alveolar cell fate selection and lifelong maintenance of AT2 cells by FGF\nsignaling. Nature Communications 13 (2022).\n10. LaCanna, R. et al. Yap/Taz regulate alveolar regeneration and resolution of lung inflammation.\nJournal of Clinical Investigation Insight 129, 2107-2122 (2019).\n11. Brownfield, D.G. et al. Alveolar cell fate selection and lifelong maintenance of AT2 cells by FGF\nsignaling. Nat Commun 13, 7137 (2022).\n12. Adams, T.S. et al. Single-cell RNA-seq reveals ectopic and aberrant lung-resident cell\npopulations in idiopathic pulmonary fibrosis. Sci Adv 6, eaba1983 (2020).\n13. Wang, Y. et al. Pulmonary alveolar type I cell population consists of two distinct subtypes that\ndiffer in cell fate. Proc Natl Acad Sci U S A 115, 2407-2412 (2018).\n14. Bingol, B. et al. The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy.\nNature 510, 370-375 (2014).\n15. Rusilowicz-Jones, E.V. et al. USP30 sets a trigger threshold for PINK1-PARKIN amplification of\nmitochondrial ubiquitylation. Life Sci Alliance 3 (2020).\n16. Tapia, P.C. Sublethal mitochondrial stress with an attendant stoichiometric augmentation of\nreactive oxygen species may precipitate many of the beneficial alterations in cellular physiology\nproduced by caloric restriction, intermittent fasting, exercise and dietary phytonutrients:\n\"Mitohormesis\" for health and vitality. Med Hypotheses 66, 832-843 (2006).\n17. Ristow, M. & Zarse, K. How increased oxidative stress promotes longevity and metabolic\nhealth: The concept of mitochondrial hormesis (mitohormesis). Exp Gerontol 45, 410-418\n(2010).\n18. Cheng, Y.W., Liu, J. & Finkel, T. Mitohormesis. Cell Metab 35, 1872-1886 (2023).\n19. Wauer, T. et al. Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and\nhydrolysis. EMBO J 34, 307-325 (2015)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Authors have adequately addressed my questions and concerns.", "reviewer_2": "The authors have addressed this reviewer's concerns.", "reviewer_3": "The authors have addressed several of the previously raised concerns. One point that would benefit from further clarification is the persistence of Krt8⁺ interstitial epithelial cells in USP30 KO mice following injury. While the authors interpret this finding as evidence of improved alveolar regeneration, alternative explanations should also be considered.\nHere the assessment was performed at day 18 post-injury, a time point at which transitional AT2–AT1 cells are typically expected to decline as repair progresses. For instance, Strunz et al. 2020 (Nature Communications)reported that Krt8⁺ transitional cells appear early after bleomycin injury, peak around day 9, and gradually decrease, becoming nearly undetectable by day 21. In addition, persistent Krt8⁺ transitional cells have been linked to dysfunctional or maladaptive repair responses, including associated to mitochondrial abnormalities and activation of the ISR, as described by Han et al. Nature 2023.\nTherefore, it would be helpful for the authors to discuss whether the sustained presence of Krt8⁺ cells might also reflect stalled or arrest differentiation of AT2 cells, rather than unequivocally improved regeneration. Additional analyses across multiple time points, as well as more detailed quantitative and qualitative characterization (e.g., via single-cell RNA sequencing), would strengthen the interpretation. Clarifying whether Krt8⁺ cells are detectable in naïve USP30 KO mice would also provide valuable context. Addressing and/or discussing these points would enhance the robustness of the conclusions regarding alveolar regeneration in the USP30 KO model."}, "author_response": "Response to the reviewers' comments:\nWe sincerely thank the reviewers and the editors for their constructive suggestions and positive feedback, which have significantly strengthened our manuscript. In the revised manuscript, we have updated the discussion as requested by the reviewer and addressed all formatting, policy, and compliance requests outlined in the editorial checklist.\nReviewer #1 (Remarks to the Author):\nAuthors have adequately addressed my questions and concerns.\nResponse: We thank the reviewer for the positive feedback and constructive suggestions,\nwhich have significantly strengthened our manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed this reviewer's concerns.\nResponse: We appreciate the reviewer’s positive feedback and insightful suggestions, which\nhave significantly improved our manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed several of the previously raised concerns. One point that would\nbenefit from further clarification is the persistence of Krt8⁺ interstitial epithelial cells in USP30\nKO mice following injury. While the authors interpret this finding as evidence of improved\nalveolar regeneration, alternative explanations should also be considered. Here the\nassessment was performed at day 18 post-injury, a time point at which transitional AT2–AT1\ncells are typically expected to decline as repair progresses. For instance, Strunz et al. 2020\n(Nature Communications) reported that Krt8⁺ transitional cells appear early after bleomycin\ninjury, peak around day 9, and gradually decrease, becoming nearly undetectable by day 21.\nIn addition, persistent Krt8⁺ transitional cells have been linked to dysfunctional or maladaptive\nrepair responses, including associated to mitochondrial abnormalities and activation of the ISR,\nas described by Han et al. Nature 2023.\nTherefore, it would be helpful for the authors to discuss whether the sustained presence of\nKrt8⁺ cells might also reflect stalled or arrest differentiation of AT2 cells, rather than\nunequivocally improved regeneration. Additional analyses across multiple time points, as well\nas more detailed quantitative and qualitative characterization (e.g., via single-cell RNA\nsequencing), would strengthen the interpretation. Clarifying whether Krt8⁺ cells are detectable\nin naïve USP30 KO mice would also provide valuable context. Addressing and/or discussing\nthese points would enhance the robustness of the conclusions regarding alveolar regeneration\nin the USP30 KO model.\nResponse: We thank the reviewer for the constructive suggestions. We agree that the\npersistence of KRT8+ transitional cells, such as those induced by ISR activation, represents\ndysfunctional or maladaptive repair, as elegantly described by Han et al. (2023)1. To directly\naddress whether KRT8+ cells in the USP30 KO mice reflect stalled differentiation or improved\nregeneration, we have analyzed the KRT8+ cells using both immunofluorescence staining and\nRNA-seq analysis at baseline and post injury. Our data support improved regeneration,\ndemonstrated by increased AT2 cell proliferation and differentiation. Notably, both KRT8+ cells\nand AT1 cells increased, indicating that the cells were not stalled in a transitional state. These\nfindings have been incorporated into the revised manuscript as Fig. S9, with relevant discussion\nadded in Lines 429-439.\n1. Krt8+ transitional cells do not spontaneously emerge in naïve USP30 KO mice.\nAs requested by the reviewer, we assessed KRT8+ transitional cells in naïve mice.\nImmunofluorescence staining of lung sections showed an absence of KRT8-expressing cells in\nthe alveolar regions of saline-treated WT and USP30 KO mice (Figure A). At baseline, KRT8\nexpression was confined to the airway epithelium, which served as an internal positive control\nfor our staining. These data show that USP30 deletion alone does not spontaneously induce a\ntransitional state.\nTranscriptomic analysis of flow-sorted Sftpc-lineage traced cells from saline-treated mice\nshowed the expression of transitional markers (Krt8, Cldn4, and Cdkn1a) was comparable\nbetween WT and Usp30ΔSftpc mice (Figure B), further confirming that no spontaneous induction\nof KRT8+ cells occurred in USP30 KO.\n2. Transcriptomic profiling suggest rapid resolution of the transitional state post injury.\nTranscriptomic analysis of Sftpc-lineage traced cells from bleomycin-injured lungs on day 14\nshowed that transitional markers (Krt8, Cldn4, and Cdkn1a) were highly upregulated in the WT\nmice, indicating induction of transitional cells. However, this induction was significantly lower in\nthe USP30 cKO mice (Figure B), suggesting AT2 cells were not stalled in the transitional state\nin the absence of USP30. Immunofluorescence staining in Figs. 2H-2R and S3D demonstrated\nUSP30 KO mice showed a significant increase in the proliferation rate of AT2 cells, as well as\nan expansion of AT2 and AT1 cell-covered areas and an increase in KRT8+ cell numbers.\nThese results suggest that enhanced AT2 cell proliferation leads to a larger pool of AT2 cells,\ncontributing to the increased numbers of KRT8+ and AT1 cells observed in USP30 KO.\nConsistent with these findings, AT2-specific USP30 KO mice also exhibited higher proliferation\nrates of AT2 cells and increased differentiation towards KRT8+ and AT1 cells. Together, these\ndata suggest USP30 deletion significantly enhances AT2 cell proliferation and differentiation,\nleading to improved regeneration.\nFigure legends: (A) Representative immunofluorescence images of KRT8 (white) and DAPI\n(blue) in the lungs of saline-treated WT and Usp30 KO mice. Scale bars: 100μm. Similar\nimmunofluorescence staining patterns were consistently observed in at least three biologically\nindependent animals per group. (B) Bulk RNA-seq analysis of Sftpc-lineage AT2 cells sorted\nfrom WTΔSftpc and Usp30ΔSftpc mice at baseline (Saline) and 14 days post-bleomycin injury. Bar\nplots show the expression levels (Transcripts Per Million, TPM) of established AT2-to-AT1\ntransitional and senescence markers, including Krt8, Cldn4, and Cdkn1a.\nReferences\n1. Han, S. et al. Mitochondrial integrated stress response controls lung epithelial cell fate. Nature\n620, 890–897 (2023)."}]

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