Fig. 4

Cellular response to oxidative stress and autophagy are activated in C2C12 cells expressing the hLMNA-R482L pathological variant. (a) GSEA enrichment plots of the Gene Ontology pathways over ranked genes between hLMNA-R482L and hLMNA-WT (p-adjusted values and normalized enrichment scores (NES) are shown). (b) Distribution of pathways from (a) among C2C12 myoblasts’ single-cell clusters using percentage of counts of bulk RNA-seq DEGs from these pathways (**p < 0.01, ***p < 0.001, ****p < 0.0001, ns—nonsignificant); Wilcoxon rank sum test was used. (c) Heatmap of normalized scaled RNA-seq counts of the upregulated DEGs in hLMNA-R482L C2C12 myoblasts found in response to oxidative stress pathway from (b). (d,e) ROS levels in myoblasts (d) and myotubes (e) using fluorescein (DCF), an indicator of ROS in living cells (n > 30, **p < 0.01, ***p < 0.001); mitochondrial DNA measured using qPCR (n > 4, NS—nonsignificant); Mann–Whitney statistical test was used. (f) Immunocytological staining of WT and R482L myoblasts treated with autophagic flux inhibitor chloroquine (CQ) shows LC3 staining enriched with LC3-II (green) isoform; F-actin (red); DAPI (blue). White arrows indicate the accumulation autophagic-related structures. (g) Western blotting for Cytosolic (LC3-I) and membrane-associated (LC3-II) isoform in R482L/WT myoblasts was used to estimate the abundance of autophagic-related structures before the degradation. Ponceau S staining served as a loading control. Uncropped blot images are in Fig. S2. (h,i) Fold changes of LC3-II (h) and LC3-I (i) proteins relative to the control band (non-treated with CQ) were determined by densitometric scanning.