Western blot analysis

Cells were lysed with RIPA lysis buffer (Applygen Technologies, Inc.). The BCA assay kit (Sangon Technology) was used to detect the protein concentration. Proteins (35 µg/lane) were subjected to 10% SDS-PAGE, and then subsequently transferred onto a PVDF membrane (EMD Millipore). Membranes were then incubated with 5% non-fat milk for 1 h at room temperature, and stained with primary antibodies against ribonucleoside-diphosphate reductase subunit M2 (RRM2; 1:1,000; cat. no. ab172476; Abcam), Bax (1:1,000; cat. no. ab32503; Abcam), Bcl-2 (1:1,000; cat. no. ab59348; Abcam), structural maintenance of chromosomes protein 4 (SMC4; 1:1,000; cat. no. HPA029449; Sigma-Aldrich; Merck KGaA), cleaved caspase-3 (1:1,000; cat. no. 9661S; Cell Signaling Technology, Inc.) and anti-GAPDH (1:1,000; cat. no. sc-32233; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Subsequently, the membrane was washed three times in TBS with 5% Tween-20, and then incubated with horseradish peroxidase-conjugated anti-rabbit (cat. no. IH-0011; 1:5,000 dilution) and anti-mouse (cat. no. IH-0031; 1:5,000 dilution) (both Beijing Dingguo Changsheng Biotechnology Co., Ltd.) antibodies for 1 h at room temperature. Finally, the membrane was visualized with Enhanced Chemiluminescence Plus reagents (Thermo Fisher Scientific, Inc.). Protein band density was normalized to the corresponding GAPDH density.