Set7-dn mutant inhibits MyoD-mediated conversion of 10T1/2 fibroblasts into myoblasts. (A) 10T1/2 fibroblasts, which stably expressed Set7 or the Set7-dn mutant, were transiently transfected with a MyoD expression plasmid. Cells were then transferred to differentiation medium for 72 h, and myogenic conversion was scored by positive staining for MHC expression (red). DAPI counter stains the nucleus. (B) The result of quantitative analysis was presented as a percentage of nuclei in myotubes from 10 randomly chosen microscopic fields. Data represent the means ± SD from at least three independent experiments. (C) 10T1/2 fibroblasts, which stably expressed Set7 or the Set7-dn mutant, were transiently transfected with a MyoD expression plasmid. Cells were then transferred to differentiation medium for 72 h, and myogenic conversion was determined by Western blot analysis for the MHC protein. The expression levels of Set7 and Set7-dn proteins are indicated. β-Tubulin was used as a loading control. (D) 10T1/2 fibroblasts were transfected with control siRNA or siRNA against Set7 together with a MyoD expression plasmid. Cells were then transferred to differentiation medium for 72 h, and myogenic conversion was scored by positive staining for MHC expression (red). DAPI counter stains the nucleus. (E) 10T1/2 fibroblasts were transfected with control siRNA or siRNA against Set7 together with a MyoD expression plasmid. Cells were then transferred to differentiation medium for 72 h, and myogenic conversion was analyzed by Western blot analysis using antibodies that recognize the indicated proteins. β-Tubulin was used as a loading control. Crtl., control. Bars, 40 µm.