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. 2015 Jul 3;12(4):5100–5108. doi: 10.3892/mmr.2015.4039

Figure 5.

Figure 5

Caspase-3 regulates the phosphorylation of Akt associated with PP2A. (A and B) A2780 and A2780/DDP cells were pre-treated with indicated concentrations of okadaic acid for 1 h and exposed to BrMC for 24 h, then detected for Akt and p-Akt (Ser473) levels by western blot analysis. β-Actin was used as a control. (C) To determine the Akt phosphorylation. A2780/DDP cells were pre-treated with or without various doses of caspase 3 inhibitor z-DEVD-fmk (20 or 80 µM) for 1 h and exposed to BrMC (5 µM) for 24 h, then p-Akt (Ser473) levels were detected using western blot analysis. (D) The apoptotic rate of the A2780 and A2780/DDP cells following pre-treatment with different doses of z-DEVD-fmk. All data are depicted graphically as the mean ± standard error of the mean of at least three independent experiments. *P<0.05, **P<0.01, vs. apoptotic rate of the A2780 and A2780/DDP cells treated with BrMC (5 µm) alone; #P<0.05, vs. treatment with BrMC (5 µm) and z-DEVD-fmk (20 µm). (E and F) Detection of the binding of Akt with PP2A. Cells were incubated with or without BrMC for 48 h, 20 µM of z-DEVD-fmk was added 1 h prior to treatment with the drug. The cells were lysed with lysis buffer for immunoprecipitation with anti-Akt antibody followed by immunoblot assay with anti-PP2A/C and anti-Akt antibodies. Data are representative of at least three independent experiments. p-Akt, phosphorylated Akt; CF, cleaved form of PP2A; PP2A, protein phosphotase 2A; BrMC, 8-bromo-7-methoxychrysin; DDP, cisplatin; z-DEVD-fmk, Caspase-3 specific inhibitor Z-Asp-Glu-Val-Asp-CH2F.