Dexamethasone (dex)

White adipocyte differentiation of hASCs was performed using the DMEM/F12-based medium as previously described according to the regularly used protocol with slight modifications to ensure successful adipocyte induction [62 ]. Adipogenic differentiation was initiated on the second day after cell confluence, followed by a 7-day induction period and a 14-day maintenance period. The induction cocktail contained 100 nM insulin (093-06351, Wako, Japan), 1 μM dexamethasone (10008980, Cayman Chemical, Ann Arbor, MI, USA), 0.5 mM IBMX (10008978, Cayman Chemical, USA), and 1 μM rosiglitazone. For the maintenance medium, 100 nM insulin and 1 μM dexamethasone were prepared with a DMEM-F12-based medium. To generate white adipocytes, hASCs were seeded at a density of 5 × 10⁴ cells per cm² and grown to confluence. Both the induction and maintenance medium was subsequently changed every 4 days for a total of 21 days.
For induced adipocytes from d-hASCs, the cells were seeded in the preadipocyte growth medium (PGM-2 ^TM Bulletkit, Lonza, Walkersville, MD, USA). When the cells reach 80% confluency, change the growth medium to adipogenic differentiation medium to induce adipogenicity. Rhinsulin, dexamethasone, IBMX, and indomethacin were added to the adipogenic differentiation medium. Refresh the medium every 10 to 12 days [14 (link)].

Brown adipocyte cell line was established by immortalization of preadipocytes obtained from interscapular BAT of mice as previously described [18 (link)]. Immortalized preadipocytes were cultured in growth medium (Dulbecco’s modified Eagle’s medium (Welgene, South Korea) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Thermo Fisher, USA)) at 37 °C in a humidified atmosphere with 5% CO₂. For adipocyte differentiation, confluent preadipocytes were exposed to differentiation medium containing 2.5 mM of isobutylmethylxanthine (IBMX, Cayman, USA), 1 μM of dexamethasone (Cayman, USA), 10 μg/mL of insulin (Sigma, USA), 125 μM of indomethacin (Cayman, USA) and 1 nM of triiodothyronine (T3, Cayman, USA) for 3 days followed by maintenance medium containing insulin (10 μg/mL) and T3 (1 nM) for 3 days.
C3H10T1/2 (ATCC, USA) cells were cultured in growth medium. For adipogenic differentiation, C3H10T1/2 cells were exposed to DMEM containing 20 ng/mL of bone morphogenetic protein 4 (BMP4, R&D systems, USA) for 2 days, differentiated in differentiation medium for 3 days, and exposed to maintenance medium for 3 days.