Low concentration of THZ1 inhibited C2C12 cell differentiation. (A)C2C12 myoblasts were induced to differentiate and meanwhile treated by gradient concentrations of THZ1 (0, 50, 100 nM). Pictures were taken three days later. (B) qRT-PCR was performed to analyze the transcript levels of muscle-specific genes including Myog, Myh3, Mylpf and MCK. RNA samples were collected one and three days after differentiation respectively. (C)MCK-luc, 3xMef-luc and Myog-luc were co-transfected with pEF-RL respectively. 24h after transfection, gradient concentration of THZ1 (0, 50, 100 nM) was added. 48h after differentiation induction, luciferase activity was measured.(D)(F) One and three days after differentiation induction, protein level of Myh3 and Myogenin were analyzed by western blot.(E) Bar graph was gray intensity analysis of figure 3D. Gray values were normalized to β-tubulin.(G) Bar graph was gray intensity analysis of figure 3F. Gray values were normalized to β-tubulin.(*P<0.05, **P<0.01, ***P<0.001, n=3, bars represent SD).