Cleaved rarp

SDS–PAGE and immunoblotting were performed according to standard procedures. Briefly, after treated with FPKc (240 µg/ml) and ES (24 µg/ml) for 0 h, 12 h, 24 h and 48 h, cells were lysed by RIPA buffer on ice. The protein samples were separated on a 10% SDS polyacrylamide gel, and then the gel was transferred to nitrocellulose membranes (Millipore, MA, USA) and blotted with primary antibodies (Caspase-3, Cleaved-RARP, Bcl-2, P53 were purchased from Cell Signaling Technology, USA) overnight at 4°C. The bound primary antibodies were then tagged with IRDye 680 Conjugated IgG (Li-cor, Biosciences) at room temperature for 1 h. And the infrared fluorescence was detected with the Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE).

Radiation treatment was performed with a 6 MV-X ray source (Varian Medical Systems, California, USA), and the cells were irradiated at a dose rate of 0.2 Gy/min. PKI-587 (MeDeCm Express, USA) was dissolved in dimethyl sulfoxide at 2 mM and stored at -20°C. Antibodies to Ki67, PI3Kp110α, PI3Kp110γ, Akt, phospho-Akt (Ser473), mTOR, phospho-mTOR (Ser2448), Bad, phospho-Bad, Caspase-3, Cleaved Caspase-3, Caspase-9, Cleaved Caspase-9, PARP, and Cleaved RARP were purchased from Cell Signaling Technology, USA. Antibodies to phospho-Rb, Rb, Cyclin D1, phospho-DNAPKcs (Ser2056), DNAPKcs, phospho-ATM (Ser1981), ATM, phospho-ATR (Ser428), and ATR were acquired from Abcam, USA. Antibodies to p70S6K, phospho-S6K1 (T421 + S424), eIF4EBP1 and phospho-eIF4EBP1 were purchased from Boster Biological Technology, China. γ-H2AX was obtained from Biolegend Biological Technology, USA. β-actin was obtained from Biosharp Life Science, China. SP-9000 was obtained from Zhongshan Gold Bridge.