Cell Culture
Lentiviruses were produced by transfecting the HEK293T cells with the core plasmids and two helper plasmids (psPAX2 and pMD2G). The transfections were implemented using the Polyethylenimine (PEI) method at the ratio of PEI : core plasmid : psPAX2 : pMD2G = 27 : 4 : 3 : 2. The medium was changed 4–6 h after transfection. After 48h, the virus-containing medium was harvested and filtered. Then, the 3T3-L1 cells were incubated overnight with the viral supernatant and 8 μg/mL polybrene. For browning differentiation, confluent 3T3-L1 cells were incubated for 2 days in a brown adipogenic induction cocktail (DMEM containing 10% FBS, 20 nM insulin, 1 nM 3,3,5-triiodo-L-thyronine (T3), 0.5 mM isobutylmethylxanthine, 0.125 uM indomethacin, and 1 mM dexamethasone). Then, the cells were kept in differentiation medium (DMEM containing 10% FBS, 20 nM insulin, 1 nM T3) for 6 days. The induction medium was changed every 2 days. At day 8, fully differentiated brown adipocytes were applied for all following experiments in this study.