For obtaining white pre-adipocites, WT and Mkk6−/− inguinal fat were mechanically and enzymatically disaggregated using type-A collagenase (2 mg/ml collagenase type-A, Roche) at 37 °C. Cell suspension passed through a 70 µm cell strainer (Falcon) to eliminate stroma and debris, and centrifuged at 400 × g for 8 min at RT. Pellet was collected and cells were counted using a CasyTon cell counter. Pre-adipocytes were inmortalized by infection with SV40T-pBABE-neo virus. Cells were differentiated to adipocytes for 9 days in 8% FCS medium supplemented with 5 μg/ml insulin, 25 μg/ml IBMX, 1 μg/ml dexamethasone, and 1 μM troglitazone. Then cultures were incubated with 100 nM T3, 100 nM T4, and 1 μM norepinephrine for 48 h before extraction. Alternatively, pre-adipocytes were differentiated to adipocytes using brown adipocyte differentiation protocol in which cells were induced to brown fat with 20 nM insulin, 1 nM T3, 125 μM indomethacin, 2 μg/ml dexamethasone, and 50 mM IBMX for 48 h, and maintained with 20 nM of insulin, and 1 nM of T3 for 8 days. In some experiments, white pre-adipocytes were infected with lentivirus containing shRNA targeting AMPK, TAK1, TAB1, p38α, or a scrambled sequence, and selected by resistance to puromycin.
Cell cultures used in this paper were tested for mycoplasma infection.
Cell cultures used in this paper were tested for mycoplasma infection.