Adipogenic Differentiation of 3T3-L1 Cells

3T3-L1 (RRID_0123) cells were maintained in high glucose DMEM (SIGMA, D5796) supplemented with 10% foetal bovine serum (FBS, Gibco, 10270), MEM non-essential amino acids, 1 mM Sodium Pyruvate (Gibco, 11360-039), 100 µg/µl penicillin and 100 µg/µl streptomycin sulphate (Gibco, 15140-122). Cells were plated at ~10% confluency and always split by 60% confluency to maintain differentiation capacity. Adipogenesis was induced two days post-confluency by replacing growth medium with induction medium containing 0.5 μM insulin (SIGMA, I1882), 1 µM dexamethasone (SIGMA, 04902), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, SIGMA, I7018), and 1 μM rosiglitazone (Abcam, Ab 120762). Induction medium was replaced after two days with maintenance medium containing growth medium supplemented with insulin and rosiglitazone only. Stable 3T3-L1 shRNA lines for knocking down of Tmem120a and Tmem120b genes were described in^{21 (link)}.