Western blotting

After the cells were treated with different concentrations of HS-173 and irradiated for various times, they were collected and washed with cold phosphate-buffered saline (PBS). The cells were then lysed with RIPA buffer containing protease and phosphatase inhibitor cocktails (GenDEPOT, Barker, TX, USA). Proteins in whole-cell lysates were resolved by sodium dodecyl sulfate protease and phosphatase inhibitor (SDS-PAGE), and transferred onto nitrocellulose membranes. The blots were first incubated with the appropriate primary antibodies, and then with horseradish peroxidase (HRP)-conjugated secondary antibodies. Antibody binding was detected using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA). Primary monoclonal antibodies against the following proteins were used: cleaved PARP, cleaved caspase-3, survivin, p-AKT, AKT (Cell Signaling Technology, MA, USA) p-Cdc2, p-ATM, ATM, p-DNA-PKcs, DNA-PKcs, p-KAP1, p-53BP1 (Abcam, Cambridge, UK) KAP1, 53BP1(Santa Cruz, Dallas, TX, USA) and β-actin (Sigma-Aldrich, OH, USA) Secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).