Fig. 5

The MEK1/2/ERK1/2 pathway is upregulated into Hrs-depleted cells and its inhibition rescues formation of myotubes. a Representative Western blotting of protein extracts from shCT and shHrs#3 C2C12 collected in Pro (lanes 1 and 7) and at 7, 24, 48, 72, and 96 h of differentiation (lanes 2–6 and 8–12) and probed with anti-HRS, -MHC, -pT202/Y204-ERK1/2, and -total-ERK1/2 antibodies. GAPDH was used as a loading control. b Quantification of the phosphorylated-ERK1/2 from similar experiments presented in a. The data are presented as a ratio of pERK1/ERK1 and pERK2/ERK2 and normalized to the Pro starting point condition. Data represent mean +/− SEM, n = 3 experiments. Significance was assessed using a two-way ANOVA test; *p < 0.05; **p < 0.01; ***p < 0.001. ns not significant. c Representative immunofluorescence images of shCT and shHrs#3 transduced C2C12 at 96 h of differentiation upon MEK1/2 inhibitor U0126 (10 μM) or DMSO treatment and stained with anti-MHC antibody as a marker of myotubes (green) and DAPI (red). Scale bar, 200 μm. d Quantification of the myogenic index corresponding to the number of nuclei present in MHC-positive structures in experiments as presented in c. Data are mean +/− SEM, each dot represents one field, n = 27 images (n = 3 experiments). Significance was assessed using a Mann–Whitney U test; *p < 0.05; ****p < 0.0001; ns not significant. e Representative Western blotting of shCT and shHrs#3 cell extracts collected at 3 days of differentiation in presence of DMSO or 10 μM of U0126 and probed with anti-pT202/Y204-ERK1/2 and -total-ERK1/2 antibodies. GAPDH was used as a loading control. f Representative immunofluorescence images of shCT and shHrs#3 transduced C2C12 at 72 h of differentiation upon treatment with either 10 μM of the U0126 MEK1/2 inhibitor or DMSO and stained with the anti-myogenin (red) and DAPI (blue) for nuclear staining. Scale bar, 40 μm. g Quantification of the myogenin-positive nuclei (%). Data represent mean +/− SEM, 10 fields have been counted per experiments, n = 2 independent experiments