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.
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.