2.1. Cell Culture and Adipocyte Differentiation

3T3-L1 cells were cultured and differentiated into mature adipocytes in six-well plates. The culture medium consisted of Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin solution. When the cell coverage reached 100%, the cells were differentiated for 2 days to produce Liquid 1, which contained DMEM, 10% FBS, 0.5 mmol/L 3-isobutyi-1-methyixanthine (IBMX), 1 μM/L dexamethasone (DXMS), and 10 μg/mL insulin. Then, the medium was changed to induce Liquid 2 for 2 days. The induced Liquid 2 contained DMEM, 10% FBS, and 10 μg/mL insulin. Cells were further cultured in DMEM and 10% FBS for 4 days in the last step of induction. The differentiated adipocytes were identified by Oil Red O staining after 8 or more days of 3T3-L1 differentiation. The accumulation of large lipid droplets was observed. The induction rate reached 90%–100%, which was suitable for the experiment. Then, we started the transfection and stimulation experiment.