Hepg2

H22, B16-F1, human hepatoma HepG2 cells and 3T3-L1 preadipocytes were purchased from China Center for Type Culture Collection (Wuhan, China). The cells were cultured in DMEM media containing penicillin/streptomycin (100 U/100 μg/ml) and 10% fetal bovine serum, and incubated at 37 °C/5% CO₂.
3T3-L1 preadipocytes were differentiated through incubation in complete DMEM containing 10 μg/mL insulin, 0.5 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) for 2 days and thereafter in complete DMEM supplemented with 10 μg/mL insulin for next 2 days. Subsequently, cells were maintained in and re-fed every 2 days with culture medium for another 4 days. Differentiation into mature adipocytes was confirmed by Oil Red O staining. Differentiated 3T3-L1 cells were cultured with complete DMEM for 24 h, and supernatants were collected as adipocytes conditional media (adi-CM).
HepG2 or B16-F1 cells were treated with adi-CM for 48 h. In the meantime, anti-TNF-α antibody (R&D system, AF-410-SP) and/or anti-IL-6 antibody (R&D system, MAB406), the inhibitor of STAT3 BP-1-102 (Selleck, S7769) or the inhibitor of NF-κB withaferin A (MCE, HY-N2065) were used at corresponding concentrations.

The human hepatoma cell line HepG2 was purchased from Cell Resource Center, IBMS, CAMS/PUMC. HepG2 cells were cultured in 10 % fetal bovine serum (FBS) (Gibco)-containing DMEM (Gibco) supplemented with 1 % NEAA (Life technologies), 1 % penicillin- streptomycin at 37 °C, 5 % (v/v) CO₂. HepG2 cells were treated with human recombinant leptin protein (R&D Systems, Minneapolis, MN, USA) at various doses (0, 5, 25, 50, 100 and 200 ng/ml) for 24 h or with 50 ng/ml leptin for various times (0, 6, 12, 24, 48 h). In other experiments, the p38MAPK inhibitor-SB203580, was administered at 10 μM, either alone or combined with 50 ng/ml leptin for 24 h. Atrovastatin (Sigma, MO) was administered at 10 μM, either alone or combined with 50 ng/ml leptin for 24 h.

The human hepatoma cell line HepG2 (American Type Culture Collection [ATCC], Manassas, VA, USA) was cultured in low glucose Dulbecco’s modified Eagle’s medium (DMEM; SH30021.01, HyClone Laboratories, Logan, UT, USA) supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin in a 5% CO₂ incubator at 37 °C. Cells were incubated in a humidified atmosphere at 37 °C and 5% CO₂ and cultured for 3 days to achieve 70% confluence before treatment with palmitate or DPP4i (teneligliptin). HepG2 cells were treated with palmitate (Sigma-Aldrich, 0.6 mM) for 18 h with or without pre-treatment with DPP4i (teneligliptin, 3 μM) for 6 h and were then analyzed. The mRNA and protein expression levels of DPP4, ER stress, and TRAIL-R2-mediated apoptosis markers were assessed. The mRNA levels of collagen1α1 and αSMA, the typical mesenchymal cell markers corresponding to the activated hepatocyte state that involves the deposition of extracellular matrix during liver fibrosis^{66 (link)}, were also analyzed. To analyze the effect of DPP4i on TRAIL-mediated apoptosis, HepG2 cells were incubated with the recombinant human TRAIL/TNFSF10 protein (TRAIL; Cat# 375-TL-010, R&D Systems) at concentrations of 50 and 100 ng/mL for 18 h, with or without pre-treatment with DPP4i (teneligliptin, 3 μM) for 6 h.