Adipocyte Differentiation Induction Protocol

White and brown adipocyte differentiation was induced as described previously, with minor modifications^{26 (link)}. For white adipocyte differentiation, cells were treated in DMEM containing 10% FBS, 10 μg/ml insulin (Wako), 2.5 μM dexamethasone (Sigma Aldrich), and 500 μM 3-isobutyl-1-methylxanthine (Sigma Aldrich). After 3 days, the medium was changed to DMEM containing 10% FBS and 10 μg/ml insulin for a further 5 days. For brown adipocyte differentiation, cells were treated in DMEM containing 10% FBS, 5 μg/ml insulin, 1 μM dexamethasone, 500 μM 3-isobutyl-1-methylxanthine, 125 nM indomethacin (Wako), 1 nM triiodothyronine (Merck), and 1 μM rosiglitazone (Wako). After 3 days, the medium was changed to DMEM containing 10% FBS, 5 μg/ml insulin, 1 nM triiodothyronine, and 1 μM rosiglitazone for a further 5 days. The medium was changed every day. After fixation by 4% paraformaldehyde/PBS, the cells were stained with Oil Red O and images were evaluated by microscopy (BZ-9000; Keyence).