Fig. 3

PI3K/Akt/mTOR signaling was activated in EOC-cis cells and BEZ235 can sensitize EOC-cis cells to cisplatin. a The protein expressions of Akt and mTOR were detected by western blot. β-actin was used as a loading control. b Combination treatment of BEZ235 and cisplatin significantly reduced the expressions of PI3K/Akt/mTOR signaling pathway proteins (p-AKT, pmTOR and p-4E-BP1) as compared to cisplatin mono-treatment group. c Colony formation ability of EOC-cis cell lines was detected after treatment with BEZ235 (½ IC₅₀), cisplatin (½ IC₅₀), or in combination for 48 h. Typical images of colony for different treatment groups are shown. d The apoptosis of EOC-cis cells after different treatments was tested with AO/EB assay. Red indicates cells undergoing apoptosis, while green indicates normal cells. Photos were obtained at 200x magnification. e The expressions of pre-apoptotic proteins in EOC-cis cells after different treatments were detected by western blot. β-actin was used as a loading control. f Quantitation analysis of ROS was performed using flow cytometry with median fluorescence intensity (MFI) detection. Fluorescence emission spectra of ROS in control and treatment groups of EOC-cis cells was shown. ROS level was significantly increased in the combination treatment group compared with single cisplatin treatment in both EOC-cis cell lines. All data were expressed as mean ± SD. ^**P < 0.01 and ^***P < 0.01 versus control group, while ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 versus cisplatin treatment group (n = 3)