Fig. 3.

Hrs silencing inhibits myoblast to myotube differentiation. a Representative Western blotting of C2C12 transduced with HIV-1 lentivectors shCT (lane 1) or shHrs-mRNA shHrs#1 (lane 2), #2 (lane 3), #3 (lane 4) and probed with the anti-HRS and anti-GAPDH. b Quantification of shCT and shHRS#3 C2C12 cell number at 6, 24, or 72 h. Data represent mean +/− SEM, n = 3 experiments. Significance was assessed using two-tailed Mann–Whitney U test. ns not significant. c Representative immunofluorescence images of shCT and shHrs#3 C2C12 myoblasts and probed with anti-Lamp1 (green) for LE/LYS or anti-EEA1 (red) for EE and DAPI (blue). White dotted squares represent a focus of the selected regions (see insets). Scale bar, 20 μm. d Representative immunofluorescence images of shCT and shHrs#1-3 C2C12 at 96 h of differentiation and probed with the anti-MHC (green) as a marker of myotubes and DAPI (red) for nuclear staining. Scale bar, 200 μm. e Representative Western blotting of shCT and shHrs#3 C2C12 myoblasts at the Pro status (lanes 1,7) or during the differentiation 7–96 h (lanes 2–6 and 8–12) and probed with the anti-HRS and -MHC. GAPDH was used as a loading control. f Quantification of the MHC protein level present in experiments like in e. Data are presented as ratio of MHC/GAPDH 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. g Quantification of the myogenic index corresponding to the number of nuclei present in MHC-positive cells as shown in d. Data represent mean +/− SEM, each dot represents one image, n = 24 images from two independent experiments. h Representative Western blotting of primary muscle cells transduced with the lentivector shCT (lane 1) or shHrs#3 (lane 2) and probed with the anti-HRS. GAPDH was used as a loading control. i Representative immunofluorescence images of shCT and shHrs#3 primary muscle cells at 48 h of differentiation and probed with the anti-MHC (green) and DAPI (red). Scale bar, 200 μm. j Quantification of the myogenic index corresponding to the number of nuclei present in MHC-positive cells as shown in i. Data represent mean +/− SEM, each dot represents one image, n = 16 images from two independent experiments