The human hepatoma cell lines HepG2.215 and HepG2-NTCP and the human normal hepatocyte cell line LO2 were used in this study, and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum (FBS) (CellCook, Guangzhou, China), 100 U/mL penicillin, and 100 mg/mL streptomycin. HepG2.215 and LO2 cell lines were provided by the Guangdong Provincial Key Laboratory of Liver Disease Research, China, as previously described [21 (link)]. The HepG2-NTCP cell line was kindly provided by Professor Liang Peng at the Third Affiliated Hospital of Sun Yat-sen University. All cells were cultured at 37°C in a humidified 5% CO2 incubator.
For ethanol and acetaldehyde treatment, hepatocytes were treated with fresh culture medium containing the indicated concentrations of ethanol and acetaldehyde for 48 h. Considering the high evaporation ability of ethanol and acetaldehyde, the medium was refreshed every 6 h. To inhibit acetaldehyde-induced reactive oxygen species (ROS), cells were treated with 10 mM N-Acetylcysteine (NAC) (MedChemExpress, NJ, USA) for 48 h. For Cycloheximide (CHX) chasing assays, cells were treated with 100 μg/mL CHX (MedChemExpress, NJ, USA) for the indicated times. To inhibit proteasomal degradation, cells were treated with 20 μM MG132 (MedChemExpress, NJ, USA) for 6 h.
HBV infection of HepG2-NTCP cells was performed as previously reported [10 ]. The HBV-containing culture medium of HepG2.215 cells were collected and concentrated polyethylene glycol precipitation (PEG) was performed. HepG2-NTCP cells were seeded in collagen-coated plates and infected with HBV (MOI 500 genome equivalents/cell) with 4% PEG 8000 and 2% dimethyl sulfoxide (DMSO) for 24 h. After incubation with HBV particles, the cells were washed three times with phosphate buffered saline (PBS) and maintained in DMEM with 10% FBS and 2% DMSO. Infected cells were incubated for at least 5 d and the medium was changed every 2 d.
For ethanol and acetaldehyde treatment, hepatocytes were treated with fresh culture medium containing the indicated concentrations of ethanol and acetaldehyde for 48 h. Considering the high evaporation ability of ethanol and acetaldehyde, the medium was refreshed every 6 h. To inhibit acetaldehyde-induced reactive oxygen species (ROS), cells were treated with 10 mM N-Acetylcysteine (NAC) (MedChemExpress, NJ, USA) for 48 h. For Cycloheximide (CHX) chasing assays, cells were treated with 100 μg/mL CHX (MedChemExpress, NJ, USA) for the indicated times. To inhibit proteasomal degradation, cells were treated with 20 μM MG132 (MedChemExpress, NJ, USA) for 6 h.
HBV infection of HepG2-NTCP cells was performed as previously reported [10 ]. The HBV-containing culture medium of HepG2.215 cells were collected and concentrated polyethylene glycol precipitation (PEG) was performed. HepG2-NTCP cells were seeded in collagen-coated plates and infected with HBV (MOI 500 genome equivalents/cell) with 4% PEG 8000 and 2% dimethyl sulfoxide (DMSO) for 24 h. After incubation with HBV particles, the cells were washed three times with phosphate buffered saline (PBS) and maintained in DMEM with 10% FBS and 2% DMSO. Infected cells were incubated for at least 5 d and the medium was changed every 2 d.
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