Palmitate

Low-passage human-derived HepG2 cells (purchased from invitrogen Carlsbad, CA, USA) were cultured in normal growth medium, Dulbecco modified Eagle’s medium (DMEM) supplemented with 4.5 mmol/L glucose, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (all purchased from Gibco, USA) in 6-well plates. To study the influence of FFA, HepG2 cells were then cultured in normal growth medium supplemented with 0.5 mM palmitate (Sangon Biotech, Shanghai, China) in 6-well plates.

To establish cellular models of lipid accumulation, primary hepatocytes were seeded in six-well plates. After cell adhesion, 0.6 mM a mixture of free fatty acids (FFAs) was added to the medium for 24 hours (at a final ratio of 2:1 with oleate and palmitate from Sangon Biotech, China). The cells were then fixed with 4% paraformaldehyde for 10 minutes and stained with nile red and Hoechst staining for 10 minute to visualize intracellular lipid accumulation. The triglyceride (TG), total cholesterol (TC) contents in isolated primary hepatocytes or liver tissue were measured using the kits from Applygen, China (#E1003 and E1005, respectively), according to the manufacturers’ instructions.