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. 2016 Dec 25;8(8):12902–12916. doi: 10.18632/oncotarget.14186

Figure 4. Superoxide mediates induction of the mitochondrial apoptotic pathway in MnSOD and GPx siRNA transfectant HUVEC cells.

Figure 4

HUVEC cells were stably transfected with scrambledsiRNA(Scr) or MnSOD si RNA and GPx si RNA. a. Western blot analysis of MnSOD and GPx protein expression (cropped) in transfected cells. β-actin served as an internal control. b. and c. transfected cells were cultured at 43°C for 2h, and incubated at 37°C for different times as indicated (0h, 0.5h, 1h, or 2h). Superoxide anion assay kit and PF6-AM analysis heat stress-induced O2.- and H2O2, respectively. d-f. Transfected cells were cultured at 43°C for 2h, then further incubated at 37°C for different times as indicated (0h, 1h, 3h, 6h, or 9h). Apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI staining. Enzymatic activity of caspase-9 and-3 was measured in cell lysates using the fluorogenic substrates Ac-LEHD-AFC and Ac-DEVD-AMC, respectively, and activity was expressed relative to the control at 37°C. Each value represents the mean ± SD of three separate experiments.