Bafilomycin

The human HCC cell lines Huh7 and Hep3B were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. These cells were cultured in DMEM (high glucose) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 100 µg/l penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The plasmids were transfected into the cells using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The ratio of the mass of nucleic acid (plasmid) to the volume of Lipofectamine 2000 was 1:2 (2 µg:4 µl), which was mixed together at room temperature for 20 min, and then added to cells for continued culture at 37°C. Stably expressed cell lines were generated using puromycin selection (2 µg/ml; Beyotime Institute of Biotechnology) for ≥7 days after 48 h transfection. Cycloheximide (CHX; Selleck Chemicals) was dissolved in dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) at a concentration of 100 mM and stored at −40°C. Huh7 and Hep3B cells were treated with 100 µM CHX for 6 h, or at different time periods, at 37°C. For protein degradation pathway analysis, Huh7 cells were treated with 1 µM proteasomal inhibitor bortezomib (BTZ; Selleck Chemicals) or 20 nM autophagy inhibitor bafilomycin (BAF; Selleck Chemicals) for 6 h at 37°C prior to western blotting.