Caspase 7

HCC cells were lysed in radioimmunoprecipitation assay buffer containing 1% proteinase inhibitor cocktail (Beijing Solarbio Science & Technology Co., Ltd.; R0020). The protein concentration was determined using a bicinchoninic acid assay. Proteins (20 µg) were loaded were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes 5% skim milk was used as the blocking buffer to incubate the membrane for 1 h at 37°C. The corresponding proteins were detected with the primary antibodies against Bcl-2 (Abcam, Cambridge, UK; cat. no. ab692; dilution: 1:500), Bax (Abcam; cat. no. ab32503; dilution: 1:2,000), caspase-7 (Bioworld Technology, Inc., St Louis Park, MN, USA; cat. no. BS6544; dilution: 1:1,000), caspase-8 (Bioworld Technology, Inc.; cat. no. AP0237; dilution: 1:1,000), PARP (Bioworld Technology, Inc.; cat. no. BS70001; dilution: 1:1,000) and GAPDH (Bioworld Technology, Inc.; cat. no. MB001; dilution: 1:1,000) (as stated in Reagents and antibodies) then incubated with a horseradish peroxide-conjugated secondary antibody (HAF008, dilution: 1:5,000; Novus Biologicals, LLC, Littleton, CO, USA), following which the proteins were visualized using an enhanced chemiluminescent substrate (WBKLS0500; Merck KGaA). Semi-quantification of the blots was conducted using ImageJ software (version no. k 1.45; National Institutes of Health, Bethesda, MD, USA).

Sorafenib (Selleck Chemicals, Houston, TX, USA) and bufalin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) were dissolved in dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) and diluted to their working concentrations (Sorafenib at concentrations of 2.5, 5, 10 µM and bufalin at concentrations of 5, 10 and 20 nM). Antibodies against Bcl-2 (Abcam, Cambridge, UK; cat. no. ab692), Bax (Abcam; cat. no. ab32503), caspase 7 (Bioworld Technology, Inc., St Louis Park, MN, USA; cat. no. BS6544), caspase 8 (Bioworld Technology, Inc.; cat. no. AP0237), PARP (Bioworld Technology, Inc.; cat. no. BS70001) and GAPDH (Bioworld Technology, Inc.; cat. no. MB001) were also used.