Fig. 3

Effect of Myostatin treatment and MSTN KO in MYOD1-induced myogenic conversion of immortalized bovine fibroblast cell line. A Experimental scheme for Fig. 3B–E. The effect of MSTN KO in MYOD1-induced myogenic conversion was assessed. B Mutation rate (i) and representative mutation patterns at the guide RNA target site (ii) after inducing knockout in bovine immortalized fibroblast cell line, integrated with Doxycycline-dependent conditional MYOD1 expression vector. PAM = protospacer adjacent motif, Dotted line = predicted cut site by Cas9 protein. (iii) Westen blot of myostatin and phosphorylated Smad3 protein from lysates of NC or MSTN KO skin fibroblasts. Relative band intensity against beta actin was compared. Data are presented as mean ± SD (n = 3). Asterisks indicate statistically significant differences (^*P < 0.05; ^***P < 0.001; n.s., not significant). C Immunofluorescence images on d 7 after treatment of 2 μg/mL doxycycline, in WT cell or cells with MSTN KO. Green = immunofluorescent stain, Blue = Nuclei. Scale bar = 150 μm. D and E Semi-quantitative analysis of myogenesis factors (D) and fusion indexes (E) on d 7 after myogenic conversion. Data are presented as mean ± SD (n = 3). Asterisks indicate statistically significant differences (^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001)