Figure 6

Myogenic differentiation analysis with immunofluorescence staining. Two-photon excitation fluorescence images of C2C12 skeletal myoblasts in (A) growth media (GM) and (B) differentiation media (DM). The cells were cultured in GM for 2 days and then cultured in GM or DM for additional 5 days. The cell nuclei were counterstained with DAPI (blue), the F-actins were stained with TRITC-labelled phalloidin (red) and the myosin heavy chains (MHCs) were stained with FITC-labelled anti-MHC antibody (green). The scale bars are 50 μm. Quantification of (C) the cell area, (D) MHC-positive area, and (E) fusion index. The fusion index was calculated as a percentage of the nuclei number in multinucleate myotubes with more than two nuclei to the total number of nuclei. Quantitative analysis was performed using ImageJ Software. The different letters in (C) and (D) denote the significant differences between each experimental group, p < 0.05. The different letters in (E) denote the significant differences between each experimental group, p < 0.05. If two groups have the same single letter (a, b, c, etc.), there is no significant difference between them. If a group is marked with a dual letter (eg, bc), it has a significant difference from the control and other groups marked with ‘a’, but does not from another group marked with ‘b’.