Cell culture and treatment

HepG2 cells were purchased from the American Type Culture Collection, and cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone; Cytiva) supplemented with 10% fetal bovine serum (FBS; HyClone; Cytiva) and 1% (v/v) penicillin/streptomycin at 37°C with 5% CO₂. The cells were stimulated with 0, 1.25, 2.5 and 5% CSE for 24 h or with 5% CSE for 6, 12 and 24 h at 37°C. The concentrations of CSE were selected according to our previous study (10). The medium was changed to DMEM containing 1% FBS during treatment with CSE and/or 5 mM ROS inhibitor [N-acetyl-L-cysteine (NAC); Sigma-Aldrich; Merck KGaA], 10 µM NF-κB inhibitor (BAY11-7082; Sigma-Aldrich, Merck KGaA), 100 µM melatonin (Sigma-Aldrich; Merck KGaA) and 2 µM SIRT1 inhibitor (Inauhzin; Sigma-Aldrich; Merck KGaA). In most experiments, HepG2 cells were pretreated with or without BAY11-7082, melatonin or Inauhzin for 1 h prior to stimulation with 5% CSE for 24 h at 37°C. However, the expression level of phosphorylated protein was measured after treatment with CSE for only 30 min, as it would be degraded if the treatment was prolonged. All experiments were performed at least three times and representative results are shown.