Primary hepatocyte isolation and culture

Mouse primary hepatocytes were isolated from 8-week-old male CTR and HuR^LKO mice. In brief, after anesthetization, the liver was perfused through the portal vein with 40 ml warm (37°C) wash buffer (0.4 g/L KCl, 0.06 g/L KH₂PO₄, 8 g/L NaCl, 0.35 g/L NaHCO₃, 0.132 g/L Na₂HPO₄.12H₂O, 0.1% glucose, 1 mM Hepes, and 0.5 mM EDTA) at 5 ml/min with the perfusate exiting through the suprahepatic vena cava, followed by digestion with 20 ml collagenase type II buffer (Gibco, #17101-015). Hepatocytes were then placed in a 10-cm Petri dish. The cell suspension was isolated using a 100-μm filter (Falcon, #352360) and centrifuged at 800 rpm for 5 min. Hepatocytes were purified with 90% Percoll (GE Healthcare Life Sciences, #17-0891-01) and washed twice. Finally, hepatocytes were counted and cultured in high-glucose DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS; BI, USA) at 37 °C in 5% CO₂, followed by starvation for 6 h before treatment with fatty acid-free BSA or 0.5 mM PA along with 1.0 mM OA for a further 24 h before harvesting. Thereafter, 0.0307 g of PA was dissolved in 3 mL 0.1 M NaOH at 75 °C for 30 min to reach a concentration of 40 mM. The solutions were then mixed with 3 mL of 40% fatty acid-free BSA in phosphate-buffered saline (PBS) at 55 °C for 30 m in yielding a final stock solution of 20 mM. OA (19.04 μL) was dissolved in 3 mL NaOH (0.1 M) at 75 °C for 30 min to reach a concentration of 20 mM. The solutions were then mixed with 3 mL of 20% fatty acid-free BSA in phosphate-buffered saline (PBS) at 55 °C for 30 min, yielding a final stock solution of 10 mM. A control BSA solution was prepared by mixing NAOH with fatty acid-free BSA. All the stock solutions were stored at 4 °C.