Human hepatoma-derived HuH-7, HepG2 and PLC/PRF/5 cells were obtained from RIKEN BioResource Center, Tsukuba, Japan, and cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Wako) supplemented with 5% fetal bovine serum (FBS; Hyclone Laboratories, Thermo Fisher Scientific, Waltham, MA, USA). The Hep3B cells were obtained from DS Pharma Biomedical, Osaka, Japan, and maintained in DMEM containing 10% FBS and MEM nonessential amino acid solution (Sigma Aldrich).
Cells were cultured with DMEM containing 5% or 10% FBS for 2 days followed by replacement with FBS-free DMEM for a further 2 days before drug treatment. GGA, other diterpenoids or other lipids (in ethanol) were dispersed in FBS-free medium, except that palmitic acid was dissolved in FBS-free medium containing 1% BSA at a final concentration of 400 μM and preincubated at 37°C for 1 h prior to treatment.
Cells were cultured with DMEM containing 5% or 10% FBS for 2 days followed by replacement with FBS-free DMEM for a further 2 days before drug treatment. GGA, other diterpenoids or other lipids (in ethanol) were dispersed in FBS-free medium, except that palmitic acid was dissolved in FBS-free medium containing 1% BSA at a final concentration of 400 μM and preincubated at 37°C for 1 h prior to treatment.