Figure 1

EE promoted C2C12 myotube hypertrophy and MyHC expression. (A) MTT assay was applied to determine the viability of undifferentiated C2C12 cells under various concentrations of EE at 24, 48, or 72 h. (B) Differentiated C2C12 cells were incubated in serum-free DMEM supplemented with 0 (CTL), 50, 100, or 200 μg/ml of EE. Representative photos were taken by a light microscope (scale bar, 100 μm) 48-h after EE treatment, which was then applied for measurement of myotube diameter (N = 90/group, data from three independent experiments). (C) The majority of C2C12 myotbe was measured around 26 to 30 μm under normal culture condition, various concentration of EE treatment significantly increased the average myotube diameter around 31 to 35 μm. (D) After 48-h treatment of vehicle (CTL), EE (50, 100, 200 μg/ml), IGF-1 (20 ng/ml), or testosterone (T, 500 nM), cells were stained with an immunofluorescent antibody against MyHC-T (green) and DAPI (blue). Scale bar, 100 μm. (E) Fusion index analysis indicated the percentage of nuclei in MyHC-positive myotube was increased after 100 μg/ml EE and IGF-1 treatment (N = 12 fields/group). (F) The abundance of MyHC expressed in C2C12 cells after 24-h treatment with vehicle (CTL), EE (50, 100, 200 μg/ml), IGF-1 (20 ng/ml), or testosterone (T, 500 nM) was evaluated by western blot assay (N = 4–6). The volume of bands stood for MyHC isoforms were digitally scanned and quantified by densitometry. After standardized with β-actin, the calculated densitometry data indicated that 24-h EE (100 and 200 μg/ml) treatment significantly increased the expression levels of MyHC-T (total, G), MyHC-F (fast type, H), and MyHC-S (slow type, I). IGF-1 and testosterone treatment served as positive controls. All data were expressed as mean ± SD. The symbol * stands for p < 0.05 as compared to CTL.