Fig. 4.

Effects of mitochondria-targeted agents on the activation of signaling pathways in HCT116 and C2C12 cells. HCT116, HCT116p53^−/− and C2C12 cells were exposed to hypoxia for different time points or treated with different mitochondria-targeted agents for indicated time and concentrations. A, immunoblot analysis of HIF1α and loading control GAPDH for the indicated cell lines. B–C, calcineurin activity was assayed by calcineurin cellular activity assay kit and nmol of phosphate released/mg of protein was plotted (means ± S.D., n = 3). D, immunoblot analysis of C2C12 cells subjected to hypoxia for HIF1α and loading control GAPDH. The blots are representative of three different runs. E, calcineurin activity, with or without treatment with mitochondria-targeted prooxidants in C2C12 cells. Activity was measured by calcineurin activity assay kit and (means ± S.D, n = 3). F, ChIP analysis was performed on a HIF1α binding site of Epo promoter to evaluate promoter occupancy by HIF1α in HCT116 and C2C12 cells after treatment with TPP+, MitoPQ for 16hr or hypoxia exposure for 12 h (n = 3 for all groups). Data represent mean ± S.D. The significance was calculated by one-way ANOVA with Tukey's multiple-comparison test, and data are presented as treatment group versus no-treatment control. In all experiments error bars represent standard deviations (*p < 0.05, **p < 0.01).