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.