Differentiation of Immortalized Brown Preadipocytes

We used an immortalized brown preadipocyte cell line (imBAT) (11 (link)), which we differentiated into mature brown adipocytes in vitro. The cells were induced at confluence for 2 days and differentiated for 2 days followed by 1 day of cultivation in standard medium [DMEM GlutaMax (Gibco), 10% fetal bovine serum (Sigma-Aldrich), 1% penicillin-streptomycin (Sigma-Aldrich)]. For induction the standard medium was supplemented with 850 nM human insulin (Sigma-Aldrich), 1 µM dexamethasone (Sigma-Aldrich, in 100% ethanol), 1 µM T3 (Sigma-Aldrich, in 1 M NaOH), 1 µM rosiglitazone (Cayman Chemicals, in 100% DMSO), 500 nM IBMX (Sigma-Aldrich, in 100% DMSO) and 125 nM indomethacin (Sigma-Aldrich, in 100% ethanol). The differentiation medium contained 1 µM T3 and 1 µM rosiglitazone. All treatments were performed on the 5^th day of differentiation. Proteasome inhibitors bortezomib (Selleckchem), epoxomicin (Millipore) and MG-132 (Calbiochem) were used at 100 nM in 100% DMSO and incubated for 6 h. For β3-adrenergic stimulation, cells were treated with 1 µM CL316,243 (Tocris, in distilled H₂O) for 6 h.