Fig. 6. Myogenic differentiation of C2C12/ß1D and C2C12/ß1A cells. (A,B) ß1D and ß1A expression during differentiation of C2C12/ß1D and C2C12/ß1A cells. (C-E) Expression of p21 (C), pRB (D) and connexin43 (E) analysed by western blotting. During differentiation, p21 is upregulated and pRB is dephosphorylated in all cell groups analysed. Connexin43 was downregulated in C2C12 and C2C12/ß1D, but not in C2C12/ß1A. (F-H,J-L) Immunostaining for MHC (green) and nuclear To-Pro3 staining (red). C2C12/ß1D and C2C12/ß1A had fewer MHC⁺ cells than C2C12 at day 3 (F-H) and day 5 (J-L) of differentiation and C2C12/ß1A myotube morphology was abnormal (H,L). (I) Percentage of MHC⁺ cells with >1 nucleus. After 3 days of differentiation, the number of C2C12/ß1D and C2C12/ß1A myotubes was less than the C2C12 control. After 5 days, the number of C2C12/ß1A myotubes is similar to the C2C12 control, in contrast to the number of C2C12/ß1D myotubes, which was still lower. (M) Fusion index (percent), being the ratio of number of nuclei in myotubes (cells with >3 nuclei) to the total number of nuclei in MHC⁺ cells. After 3 and 5 days of differentiation, both C2C12/ß1D and C2C12/ß1A myotubes have fewer nuclei than C2C12 myotubes. Bars represent means ± s.d. *P0.05; **P0.01.