Hepg2 cells

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.

HepG2 cells were obtained from Sangon Biotech Co., Ltd. (D611027-0001, Shanghai, China) and cultured in Dulbecco’s modified Eagle medium (DMEM) medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 KU/L penicillin (Gibco, Carlsbad, CA, USA) and 100 μg/mL streptomycin (Gibco, Carlsbad, CA, USA). Cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) (purity~98%) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) (purity~99%) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Hifair^® II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) and Hieff^® qPCR SYBR Green Master Mix (Low Rox) were purchased from Yeasen Biotechnology Co., Ltd. (Shanghai, China).
HepG2 cells were incubated overnight in 96-well plates at a density of 1 × 10⁶ cells/well. The cells were cultured with OA and PIP at different concentrations for 24 h. Subsequently, the final concentration of 0.5 mg/mL MTT was added and incubated at 37 °C for 4 h. After removing the MTT from each well, the insoluble formazan crystals were dissolved by DMSO, and the absorbance was measured with a microplate reader (PerkinElmer, Waltham, MA, USA) at 490 nm.