Fig. 3: MuSC senescence is a hallmark of DM1 in vitro and in situ.

a Representative micrographs of control and DM1 myoblasts stained for FITC SA-ß-Gal (green). Scale bars: 300 μm. b Quantification of the percentage of SA-ß-Gal+ senescent cells (n = 4 ctrl and 6 DM1 cell lines; average of 467 cells [257-705 cells] counted per sample) **p = 0.0023 (Two-tailed unpaired T-Test with Welch’s correction). c qPCR for the senescence markers P16 and P21 in control and DM1 myoblasts (n = 3 ctrl and 4 DM1 cell lines); *p = 0.046 (two-way ANOVA followed by Bonferroni’s multiple comparisons). d Growth curve of control and DM1 myoblasts cultured for 7 days (n = 5); *p = 0.042 (Two-tailed multiple unpaired T-test). e Representative micrograph of co-immunostaining for RNA FISH (CUG repeats, red), SA-β-Gal (green), and DAPI (blue). Red lines identify a senescent cell and gray dashed lines a non-senescent cell. Scale bar: 46.2 μm. f Quantification of the number of intranuclear RNA foci per senescent or non-senescent cell in DM1 myoblasts. (n = 6 samples; average of 50 cells [22-104 cells] counted per sample) **p = 0.0017 (Two-tailed unpaired T-test). g Co-immunofluorescence labeling of PAX7 (green) and P16 (magenta) on control and DM1 muscle sections. White arrowheads indicate PAX7 + P16- MuSCs and white arrows indicate PAX7 + P16+ senescent MuSCs. Scale bars: 50 μm. h Quantification of senescent MuSCs expressing PAX7 and P16 (n = 3 Ctrl and 5 DM1 biological samples; average of 110 cells [23-246 cells] counted per sample) *p = 0.0219 (Two-tailed unpaired T-Test with Welch’s correction). i qPCR for the senescence markers P16 and P21 on control and DM1 muscle biopsies (n = 5 Ctrl and 9 DM1 biological samples). **p = 0.0075 (P16) and **p = 0.004 (P21) (two-tailed Mann–Whitney test). j Schematic showing the experimental design of the conditioned medium (CM) assay. Created with BioRender.com. k Cell proliferation of healthy myoblasts treated with control or DM1 CM for 4 days (n = 4); *p = 0.0452 (Two tailed unpaired T-test with Welch’s correction). l Representative micrographs of myotubes differentiated for 3 days and then cultured with control or DM1 CM for 2 days (MyHC in green; DAPI in blue). Scale bars: 50 μm. m Fusion index of myotubes treated with control or DM1 CM. (n = 4 Ctrl and 3 DM1 biological samples; average of 860 nuclei cells [576-1376 cells] counted per sample). *p = 0.0219 (two-tailed unpaired T-test). Data are expressed as means ± SEM. Source data are provided as a Source Data file.