Protein Extraction and Western Blot Analysis

Cells and tissues were lysed in RIPA buffer. Tumors were ground in liquid nitrogen and lysed. Protein concentration was determined using the BCA Kit (Beyotime Institute of Biotechnology). Proteins were mixed with loading buffer and heated at 70°C for 10 minutes on sodium dodecyl sulfate (SDS)-polyacrylamide gels at 30 μg per lane. The proteins were transferred to polyvinylidene fluoride (PVDF, Millipore, MA, USA) after electrophoresis. Membranes were blocked for 2 hours in 5% BSA and incubated overnight at 4°C with antibodies against γ-H2AX, ATM, ATR, Chk1, cell-cycle controller-2 (Cdc2), E-cadherin, vimentin, caspase-3, and caveolin-1 (Cav-1). The blots were then incubated with HRP-conjugated secondary antibody (1:1000; Santa Cruz Biotechnology). Finally, bands were visualized by enhanced chemiluminescence (Thermo Scientific Pierce, IL, USA).