Anti topoisomerase 2 α

Compound-treated DU145 cells were lysed with RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing 1% protease inhibitor cocktail (GenDEPOT, Katy, TX, USA). The lysed cells were incubated on ice for 20 min and centrifuged at 4 °C for 20 min at 12,000 rpm to obtain the supernatant. The protein was quantitated using a Pierce^TM BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Samples were mixed with 2× loading buffer (1 mM Tris-HCl, pH 6.8, glycerol, 10% SDS, bromophenol blue, beta-mercaptoethanol) and heated at 98 °C for 3 min. Samples were separated by SDS-PAGE and transferred to a PVDF membrane, and the membrane was incubated overnight with primary antibodies at 4 °C. Anti-topoisomerase IIα (#12286), anti-Cyclin D1 (#2922), anti-cleaved-PARP (#9541), anti-Bax (#2772), anti-Bcl-2 (#2872), anti-β-actin (#4967) and α-Tubulin (#2144) were from Cell Signaling Technology (Danvers, MA, USA). Anti-p27^kip1 (#E11-0965B) was from EnoGene Biotech Co, Ltd. (NY, USA). The images were scanned by LAS-3000 (Fuji Photo Film Co., Ltd., Tokyo, Japan) and analyzed using Multi-Gauge Software (Fuji Photo Film Co., Ltd., Tokyo, Japan).