MEF culture, differentiation, and treatment

Primary Mouse embryonic fibroblasts (MEFs) were generated from pregnant mice at day 13.5 post coitum as described before (Braga et al. 2013). The embryonic head and internal organs were removed, the remaining carcasses were rinsed in 1× PBS and minced with scissors. Next, minced carcasses were suspended with 20ml 0.025% trypsin/EDTA (Roche) in a 50ml EP tube, and incubated in a water bath for an hour at 37°C. The tube was shaken every 15 min. The trypsin was neutralized by adding DMEM with 10% FBS, then passed through a 100μm nylon mesh cell strainer into a new tube to remove undigested tissues. Cell suspensions were centrifuged and resuspended in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. The cells were plated into a 10cm dish and cultured until confluent. Two days after confluence (day 0), the medium was changed to preadipocyte differentiation medium (DMEM supplemented with 10% FBS, 3-isobutyl-1-methylxanthine (0.5 mM, I5879, Sigma-Aldrich), insulin (10 μg/ml, 1342106, Sigma-Aldrich), dexamethasone (2 μM, D1756, Sigma-Aldrich), and rosiglitazone (2.5 μM, R2408, Sigma-Aldrich). From day 2, medium containing 10 μg/mL insulin and 2.5 μM rosiglitazone was changed every 2 days. The cells were divided into two groups and were infected with adenovirus on day 6 and the culture medium was changed to 2% (w/v) fatty acid-free BSA on day 8. Cells were treated with or without 8-Br-cAMP (1 mM, B5386, Sigma) for 4 and 12 h after 24 h pre-incubation with Oltipraz (10 μM, HY-12519, MCE). All cells were maintained in a 37°C and 5% CO2 humidified atmosphere.