Figure 4

SHME protects against H ₂ O ₂ -induced DNA damage in C2C12 cells. C2C12 cells were pretreated with 300 μg/ml SHME for 1 h and then incubated with and without 1 mM H₂O₂ for 6 h. (A) To detect cellular DNA damage, the comet assay was performed and representative pictures of the comets were taken using a fluorescence microscope at × 200 original magnification. (B) To quantify the degree of apoptosis, the cells were stained with Annexin V, and the percentages of apoptotic cells were then analyzed using flow cytometric analysis. Each point represents the means of two independent experiments. (C) Whole-cell lysates were prepared and subjected to Western blot analysis with a specific antibody against phospho-histone γH2A.X. Actin was used as the loading control. A representative blot from three independent experiments is shown. The numbers represent the average densitometric analyses as compared with actin in, at a minimum, two or three different experiments.