Cell culture and small interfering RNA (siRNA) transfection

CRC LoVo cells were obtained from the Cell Bank of the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were grown in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific Inc.), penicillin (100 U/ml) and streptomycin (100 µg/ml) in a humidified atmosphere at 37°C with 5% CO₂. Cells were periodically detached with 0.25% trypsin-ethylene diamine tetraacetic acid solution and subsequently replated. Cells in the logarithmic phase were used for the experiments. Livin was knocked down with siRNA against livin (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Duplex sequences of siRNA-livin were as follows: a-GGAGUUGCGUCUGGGCUCCUCUAU and b-AUAGAAGGAAGGCCAGACGCAACUCC. The transfection mixture consisted of antibiotic-free culture medium and 100 nM siRNA. The non-targeting scrambled siRNA (Invitrogen; Thermo Fisher Scientific, Inc.) was used as a negative control. Following siRNA transfection according to the manufacturer's protocol, the cells were incubated and then used for various analyses.