Fig. 3

Constitutive hyperactivation of IL6/STAT3 signaling in Cav3 mutant myotubes. a Immunoblot analysis of pSTAT3 and STAT3 levels in WT, Cav3 P28L, and Cav3 R26Q myotubes stimulated for the indicated times with 10 ng mL⁻¹ IL6. Tubulin serves as a loading control. b Quantification of STAT3 activation of a, corresponding to the ratio pSTAT3 on STAT3 total levels after normalization to tubulin levels. c Confocal microscopy of immunofluorescent pSTAT3 in WT, Cav3 P28L, and Cav3 R26Q myotubes stimulated or not for 15 min with 10 ng mL⁻¹ IL6. White dashed lines outline nucleus boundaries. d Quantification of pSTAT3 nuclear translocation in c corresponding to nuclei/cytoplasm mean intensity ratio of pSTAT3. e Immunoblot analysis of pSTAT3 levels in WT ctl (siCtl) and Cav3-depleted (siCav3) myotubes stimulated for the indicated times with 10 ng mL⁻¹ IL6. f Quantification of STAT3 activation in e, corresponding to the ratio pSTAT3 on STAT3 total level after normalization with tubulin level. g Expression of STAT3 related genes: from left to right SOCS3, MYH8, ACTC1, and ACTN2 in WT, Cav3 P28L, or Cav3 R26Q myotubes. c Scale bar = 10 µm. Reproducibility of experiments: a, c, e Representative data. b Quantification was done on 4 independent experiments. d Quantification was done on 3 independent experiments (0 min: WT n = 41 cells, P28L n = 25 cells, R26Q n = 21 cells; 15 min: WT n = 22 cells, P28L n = 30 cells, R26Q n = 30 cells). f Quantification was done on 4 experiments. g Quantification was done on 5 (SOCS3), 8 (MYH8), 3 (ACTC1), and 7 (ACTN2) independent experiments. Mean value ± SEM. b, f Statistical analysis with two-tailed paired t test. d, g Statistical analysis with two-tailed unpaired t test *P < 0.05; **P < 0.01; ***P < 0.001; ns non-significant