Set7 affects skeletal muscle myoblast proliferation and differentiation. (A) C2C12 myoblasts were transfected with Set7 or a dominant-negative mutant of Set7 (Set7-dn) together with a GFP expression construct. Cells were transferred to differentiation medium for 72 h, and myoblast differentiation was observed by fluorescent microscopy. The quantitative analysis result of the mean myotube numbers from 10 randomly chosen microscopic fields was presented on the right. Data represent the means ± SD from at least three independent experiments. Bar, 30 µm. ***, P < 0.001. (B) C2C12 myoblasts were transfected with Set7 or Set7-dn. Cells were transferred to differentiation medium for the indicated time, and the expression of myogenic differentiation genes was assayed by RT-PCR. GAPDH was used as a loading control. (C) C2C12 myoblasts were transfected with Set7 or Set7-dn. Cells were transferred to differentiation medium for the indicated time, and expression of myogenic differentiation proteins was assayed by Western blot analysis. β-Tubulin was used as a loading control. (D) C2C12 myoblasts were transfected with Set7 or Set7-dn. Cells were transferred to differentiation medium for the indicated time, and myocyte proliferation and differentiation were assayed by immunostaining with the indicated antibodies. Phos H3, phospho–histone H3; MHC, myosin heavy chain. Bar, 20 µm. (E) The MCK promoter luciferase reporter (MCK-Luc) was cotransfected with a MyoD expression plasmid together with an increasing amount of Set7 or the Set7-dn expression plasmid. Luciferase activity was determined 48 h after transfection and was presented as relative luciferase activity in which the control was assigned a value of 1. Data represent the means ± SD from at least three independent experiments in duplicate. SK, skeletal muscle; MCK, muscle creatine kinase; D0, day 0. *, P < 0.05; **, P < 0.01.