Cell culture and adipocyte differentiation

3T3-L1 mouse embryonic preadipocyte cells were purchased from the American Type Culture Collection (CL-173; ATCC, Manassas, VA, USA). 3T3-L1 preadipocytes were passaged in growth medium (GM; high glucose DMEM supplemented with 10% calf serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin), with a change of medium every 2 days, in a humidified atmosphere of 5% CO₂ at 37°C. To induce adipocyte differentiation (S1 Fig), 3T3-L1 preadipocytes were cultured until post-confluence (Day 0), at which point the GM was replaced with fresh differentiation medium (DM; high glucose DMEM with 10% FBS including MDI (isobutylmethylxanthine (IBMX, 0.5 mM), dexamethasone (1 μM), and insulin (1 μg/mL))) for 2 days in the presence of spiramycin. On day 2 (Day 2), the medium was replaced with DM (high glucose DMEM with 10% FBS containing 10 μg/ml insulin only). After 2 days (Day 4), the cells were re-fed with fresh high-glucose DMEM with 10% FBS without MDI. On day 6 (Day 6), fully differentiated cells were analyzed for each experiment. A schematic representation of the procedure is presented in S1A Fig.