Figure 2: miR-431 promotes myogenic differentiation by targeting Pax7.

(a) The differentiation of C2C12 cells stably overexpressing miR-431 was examined by staining for MyoG after 12 h of culture in DM. Cells overexpressing miR-1 served as the positive control and cells with an empty vector as the NC. Scale bar, 50 μm. (b,c) MyoG expression in the C2C12 cells described in a was analysed using semiquantitative RT–PCR (b) and western blot analysis (c). GAPDH was the loading control. (d)The differentiation of C2C12 cells stably overexpressing miR-431 was examined by staining for MHC after 36 h of culture in DM. Cells overexpressing miR-1 served as the positive control and cells with an empty vector as the NC. Scale bar, 50 μm. (e,f) The expression of MHC in C2C12 cells in d was analysed using semiquantitative RT–PCR (e) and western blot analysis (f). GAPDH was the loading control. (g) Pax7 was overexpressed in C2C12 cells stably overexpressing miR-431 and NC cells. Differentiation of C2C12 cells was examined by staining for MHC after 24 h of culture in DM. Scale bar, 50 μm. (h) MHC⁺ cells were calculated based on staining described in panel g. (i) MHC expression in the cells in g detected using real-time qRT–PCR. GAPDH was the internal control. Error bars represent means±s.e.m. of three independent experiments. **P<0.01, ***P<0.001. Two-tailed Student’s t-test was used for all statistical testing.