Journal: bioRxiv

Article Title: Loss of Parkin Disrupts Nuclear and Mitochondrial Programs Required for Muscle Regeneration

doi: 10.64898/2026.03.20.712989

Figure Lengend Snippet: A-C) Parkin transcript and protein levels in FACS-purified MuSCs from MuSC Park2 +/+ and MuSC Park2 -/- mice 1 week after the last tamoxifen treatment ( n =4 mice per group). Protein abundance was quantified as the total volume of PARKIN + structures (B) in cells labeled with antibodies against PARKIN and TOM20 ( n =11-70 cells from 2-4 mice per group,) (C). D-E) transcript abundance expressed in Transcript Per Million (TPM) for genes involved in Parkin- and Receptor-mediated mitophagy ( n =3 mice per group). F-G) Volume of mitochondria colocalized to autophagosomes expressed in absolute value or relative to total mitochondrial volume in freshly sorted quiescent and 4h in vitro activated MuSCs ( n =41-52 cells from 3 mice in each group). H) Confocal image and 3D reconstruction of TOM20-labeled mitochondria (green) and LC3-labeled autophagosomes (red) in each of the experimental conditions examined. The volume of mitochondria overlapping with autophagosomes is shown in yellow in the right-end panels, where mitochondria and autophagosomes surfaces have been removed to highlight changes in colocalization. All data are presented as mean ±SEM. ns: not significant, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0001 on unpaired two-tailed t tests or one-way ANOVAs.

Article Snippet: Park2 loxP/loxP mice were obtained from Lexicon Pharmaceuticals and bred with Pax7CreERT2 GAKA mice obtained from Jackson’s laboratory ( B6.Cg-Pax7 tm1(cre/ERT2)Gaka /J ) to develop a Tamoxifen inducible and conditional knockout of PARKIN in PAX7 + adult MuSCs.

Techniques: Purification, Quantitative Proteomics, Labeling, In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Parkin Disrupts Nuclear and Mitochondrial Programs Required for Muscle Regeneration

doi: 10.64898/2026.03.20.712989

Figure Lengend Snippet: A) Representative H&E stains of fibers in contralateral uninjured and injured TA muscle of MuSC Park2 +/+ and MuSC Park2 -/- mice 21 days after CTX injury. B) Mean cross sectional area (CSA) of fibers in contralateral (Cl) uninjured and injured TA muscle at 4, 7 14- and 21-days post CTX injury ( n =6-21 ROIs from 3-5 TA muscle per group). C) Fiber size distribution of uninjured and injured muscles at 21 days post injury. D) Representative images of PAX7 + and DAPI stained TA cross sections in uninjured and injured TA 21 days post CTX injury. E) Number of PAX7 + positive cells in uninjured and injured TA muscles at 4, 7 14 and 21-days post CTX injury. Given the differences observed in fiber CSA between genotypes, cell count is expressed as the number of PAX7 + cells divided by the number of fibers present in each 500 µm 2 ROI analyzed ( n =5-15 ROIs from 3-5 TA muscle per group). F) Representative images of PAX7 and MYOD labeled MuSCs at the surface of EDL fibers after 1 and 48h of culture. G) Proportion of quiescent (PAX7 + only) and activated MuSCs (PAX7 + /MYOD + ) and MYOD + only at 1, 24 and 48 hours post isolation ( n =65-78 cells from 13-16 fibers isolated from 3-4 mice per genotype). H) Representative images of PAX7 and MYOG labeled MuSCs at the surface of EDL fibers after 96h of culture. I) Proportion of MuSCs expressing MYOG after 96h in culture (n=634-712 MuSCs from 4-5 fibers isolated from 3 mice per genotype). All data are presented as mean ± SEM. ns: not significant, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0001 on unpaired two-tailed t tests or one-way ANOVAs.

Article Snippet: Park2 loxP/loxP mice were obtained from Lexicon Pharmaceuticals and bred with Pax7CreERT2 GAKA mice obtained from Jackson’s laboratory ( B6.Cg-Pax7 tm1(cre/ERT2)Gaka /J ) to develop a Tamoxifen inducible and conditional knockout of PARKIN in PAX7 + adult MuSCs.

Techniques: Muscles, Staining, Cell Characterization, Labeling, Isolation, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Parkin Disrupts Nuclear and Mitochondrial Programs Required for Muscle Regeneration

doi: 10.64898/2026.03.20.712989

Figure Lengend Snippet: A) ATP levels in freshly isolated MuSCs from MuSC Park2 +/+ and MuSC Park2 -/- mice measured in absence and presence of the ATP synthase inhibitor Oligomycin (20 µM). B) Contribution of OXPHOS to cellular ATP levels calculated as the difference in ATP levels in absence and presence of Oligomycin ( n =4 MuSC sorts per genotype). C) Mean TMRE fluorescence intensity in freshly isolated MuSCs expressed as fold change of control( n =3-4 MuSC sorts per genotype). D) Average volume of individual mitochondria per cell in freshly isolated MuSCs labeled with TOM20 (n=48-52 cells from from 3 mice in each group). Data was obtained following confocal imaging and 3D reconstruction. E) Proportion of quiescent (PAX7 + only) activated (PAX7 + /MYOD + ) and committed (MYOD + only) MuSCs in EDL fibers cultured in absence or presence of 10 µM mitoTEMPO for 24h (n=52-135 MuSCs from 5 fibers from 4 mice per genotype). F) Transcript levels expressed in Transcript Per Million (TPM) for key genes regulating mitochondrial biogenesis and mtDNA replication in freshly isolated MuSCs (n=3 mice per genotype). All data are presented as mean ± sem. nd: not different, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0001 on unpaired two-tailed t tests or one-way ANOVAs comparing MuSC Park2 +/+ and MuSC Park2 -/- . G) Venn Diagrams presenting the number of mitochondrial genes (MitoMiner mitochondrial localisation database ) that are differentially enriched in freshly isolated PARKIN deficient MuSCs compared to controls. Fold enrichment of mitochondrial genes is shown below Venn diagram along with the hypergeometric test p value. H) g:Profiler enrichment map illustrating the main mitochondrial processes downregulated in PARKIN deficient MuSCs compared to controls. The auto-annotation tool was used on Cytoscape to automatically generate cluster labels.

Article Snippet: Park2 loxP/loxP mice were obtained from Lexicon Pharmaceuticals and bred with Pax7CreERT2 GAKA mice obtained from Jackson’s laboratory ( B6.Cg-Pax7 tm1(cre/ERT2)Gaka /J ) to develop a Tamoxifen inducible and conditional knockout of PARKIN in PAX7 + adult MuSCs.

Techniques: Isolation, Fluorescence, Control, Labeling, Imaging, Cell Culture, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Parkin Disrupts Nuclear and Mitochondrial Programs Required for Muscle Regeneration

doi: 10.64898/2026.03.20.712989

Figure Lengend Snippet: A) g:Profiler enrichment map illustrating nuclear processes downregulated in PARKIN deficient MuSCs compared to controls. The auto-annotation tool was used on Cytoscape to automatically generate cluster labels. B) Confocal image and 3D reconstruction of mitochondria (TOM20, green), nucleus (DAPI, blue) and PARKIN + foci (red) in freshly isolated and 24-48h in vitro activated MuSCs from wild type mice. PARKIN + foci located within the nuclear compartment is shown in Yellow in the right-end panels, where mitochondria and nuclear surfaces have been removed for clarity. C) Quantification of cytosolic PARKIN + foci in in situ fixed, freshly sorted quiescent and 24-48h in vitro activated MuSCs. PARKIN was defined as cytosolic if the overlap ratio between PARKIN + foci and nuclear volume ranged between 0 and 0.4. D) Quantification of nuclear PARKIN + foci in in situ fixed, freshly sorted quiescent and 24-48h in vitro activated MuSCs. PARKIN was defined as nuclear if the overlap ratio between PARKIN + foci and nuclear volume was above 0.8 ( n =10-36 cells from 4 mice per genotype and time point). In panel C and D, note the absence of PARKIN staining in MuSC Park2 -/- cells confirming the specificity of the staining. E) Quantification of Ubiquitin + foci within the nuclear compartment in MuSC Park2 +/+ and MuSC Park2 -/- MuSCs at 18hr into culture. ( n =24-32 cells from 3 mice per genotype) F) Confocal image and 3D reconstruction of K63-Ubiquitin + foci (red) and nucleus (DAPI blue) and nuclear ubiquitin (yellow) in 18hr cultured MuSCs. Ubiquitin signal was defined as nuclear if the overlap ratio between Ubiquitin + foci and nuclear volume was above 0.8. G) Confocal image and 3D reconstruction of PARKIN (red), SC35/SRRM2 + speckles (green) and nucleus (DAPI, blue) in freshly isolated wild-type MuSCs. H) Proportion of nuclear speckles labeled by PARKIN in freshly isolated wild type MuSCs. ( n =64 cells from 6 mice) I-J) Quantification of total speckle content per nucleus and volume of individual speckles in MuSC Park2 +/+ and MuSC Park2 -/- MuSCs. ( n = 23-24 cells from 3 mice per genotype). K) Confocal image and 3D reconstruction of speckles (SC35/SRRM2, green) and nucleus (DAPI, blue) in freshly isolated MuSC Park2 +/+ and MuSC Park2 -/- MuSCs. All data are presented as mean ± SEM. ns: not significant, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0001 on unpaired two-tailed t tests or one-way ANOVAs comparing MuSC Park2 +/+ and MuSC Park2 -/- .

Article Snippet: Park2 loxP/loxP mice were obtained from Lexicon Pharmaceuticals and bred with Pax7CreERT2 GAKA mice obtained from Jackson’s laboratory ( B6.Cg-Pax7 tm1(cre/ERT2)Gaka /J ) to develop a Tamoxifen inducible and conditional knockout of PARKIN in PAX7 + adult MuSCs.

Techniques: Isolation, In Vitro, In Situ, Staining, Ubiquitin Proteomics, Cell Culture, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Parkin Disrupts Nuclear and Mitochondrial Programs Required for Muscle Regeneration

doi: 10.64898/2026.03.20.712989

Figure Lengend Snippet: A) Transcriptomic landscape of PARKIN-deficient quiescent MuSCs. Left: Distribution of affected genes by type of alteration—differential expression only (DE only, blue), differential transcript usage only (DS only, tan), or both (DE+DS, white; total n=1,188 genes). B) Directionality of non-productive transcript changes. Among genes with IR, NMD, or processed transcript isoforms (n=384 events), stacked bar indicates the proportion where these non-productive isoforms are upregulated in Park2 -/- (green, n=99), downregulated in Park2 -/- (blue, n=186), or show bidirectional changes with both up- and downregulated non-productive isoforms (white, n=99). C-E) Gene Ontology enrichment analysis stratified by directionality of non-productive transcript changes. Bubble plots show enriched biological processes (GO:BP), cellular components (GO:CC), and Reactome pathways. (C) Enrichment analysis of genes with downregulated non-productive isoforms (n=186). (D) Enrichment analysis of genes with bidirectional changes showing both upregulated and downregulated non-productive isoforms (n=99). (E) Enrichment analysis of genes with upregulated non-productive isoforms (n=99), revealing selective enrichment for RNA splicing, mRNA processing, and spliceosomal machinery pathways. F) Isoform switching in core spliceosome and nuclear speckle genes. For 22 representative splicing machinery genes, log2 fold-change ( Park2 -/- vs Park2 +/+ ) of individual transcript isoforms grouped by whether genes show both differential expression and splicing (DE+DS, top) or splicing changes only (DS Only, bottom). The predominant pattern shows coordinated downregulation of protein-coding transcripts concurrent with upregulation of non-productive variants.

Article Snippet: Park2 loxP/loxP mice were obtained from Lexicon Pharmaceuticals and bred with Pax7CreERT2 GAKA mice obtained from Jackson’s laboratory ( B6.Cg-Pax7 tm1(cre/ERT2)Gaka /J ) to develop a Tamoxifen inducible and conditional knockout of PARKIN in PAX7 + adult MuSCs.

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Loss of Parkin Disrupts Nuclear and Mitochondrial Programs Required for Muscle Regeneration

doi: 10.64898/2026.03.20.712989

Figure Lengend Snippet: A) Number of MuSCs per cluster in EDL fibers from MuSC Park2 +/+ and MuSC Park2 -/- mice after 48 and 72h in culture (n=83-162 clusters from 4-7 fibers isolated from 3 mice per genotype). B) Number of MuSCs per cluster in EDL fibers from Pink1 +/+ and Pink1 -/- mice after 48 and 72h in culture in the Cairns et al. study . C) Representative images of MYOD / Ki67 / EdU immunostainings in EDL fibers. Arrow colors illustrate the various cell cycle states (proliferating, non-proliferating, premature exit, G1/S delay). D) Proportion of MYOD + MuSCs found in each the cell cycle state at 48 and 72h in culture. E) MuSC cluster size in EDL fibers cultured in absence or presence of 10 µM mitoTEMPO for 24h (n=52-135 MuSCs from 5 fibers from 4 mice per genotype). All data are presented as mean ± SEM. ns: not significant, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0001 on one-way ANOVAs.

Article Snippet: Park2 loxP/loxP mice were obtained from Lexicon Pharmaceuticals and bred with Pax7CreERT2 GAKA mice obtained from Jackson’s laboratory ( B6.Cg-Pax7 tm1(cre/ERT2)Gaka /J ) to develop a Tamoxifen inducible and conditional knockout of PARKIN in PAX7 + adult MuSCs.

Techniques: Isolation, Cell Culture