Figure 3.

Stimulation of myogenic differentiation by TM-53 and TM-54. C2C12 cells were induced to differentiate into mature myocytes in the presence of vehicle, TM-53 (10 μM) or TM-54 (10 μM) for 6 days. (A) Differentiated myocytes were fixed and incubated with MyHC Ab, followed by counterstaining with DAPI and observation under a fluorescence microscope. (B) For quantitative analysis, the MyHC-expressing cells were counted from six different regions of the three independent culture experiments. (C) Multinucleated MyHC+ cells (between 50 and 100 cells counted) were scored in 5 different fields of view per sample. Fusion index (>2 nuclei per cell) was calculated by dividing the number of multinucleated cells by the number of MyHC+ cells from three independent experiments. (D) Total cell extracts were harvested at the indicated time points and subjected to immunoblotting with anti-MCK Ab. (E) Total RNA was prepared from differentiating cells and the relative expression of MyHC was determined by real-time PCR analysis. The results of (D) and (E) are given as mean ± SEM for three experiments. (F) Cells were harvested at day 5 and incubated with Fura-2AM for 1 h. Cells were washed with calcium-free buffer and measured at 340 and 380 nm. The ratio of calcium influx was calculated at the indicated times. This experiment was repeated three times and a representative graph is shown. *P < 0.05; **P < 0.005; ***P < 0.0005; significantly different from vehicle.