Figure 5.

TAZ-dependent stimulatory effect of TM-53 and TM-54 on myogenic differentiation. (A–C) Control (Coni) and TAZ knockdown (TAZi) C2C12 cells were established and differentiated into myocytes in the presence of TM-53 or TM-54. Cell extracts were harvested at day 6 and subjected to immunoblotting with anti-MyHC antibody. MyHC expression level was analysed using densitometry (A). Total RNA was collected at day 6 and used for reverse transcription and real-time PCR analysis. Relative expression level of MCK, Myog and TAZ was calculated after normalization to β-actin levels. *P < 0.05; **P < 0.005; ***P < 0.0005; significantly different from vehicle-treated group; #P < 0.01; ##P < 0.001; significantly different from coni group (B). Cells were harvested at different time points and used for MCK activity assay using MCK colorimetric assay kit (BioVision Inc.). MCK activity was determined as nmoL·min⁻¹·mL⁻¹ (C). (D) Highly transfectable 293T cells were transfected with Flag-TAZ and MyoD expression vector and were treated with TM-53 (10 μM) and TM-54 (10 μM) for 24 h. Cell extracts were subjected to immunoprecipitation and immunoblotting. (E) WT and KO MEF cells were transfected with MyoD expression vector together with either pMyog-luc or pMCK-luc. Cells were then treated with TM-53 (10 μM) and TM-54 (10 μM) for 24 h and collected for reporter gene assay. The promoter activity was expressed as fold change after normalization with β-galactosidase activity. The MyoD-induced promoter activity in vehicle-treated cells was set as 1. All experiments in Figure 5 were repeated at least three times and data are given as mean ± SEM. Representative image from three blots was shown in Figure 5A and D.