2.2. hMADS cell culture

The establishment and characterization of hMADS cells has been described [36], [37], [38], [39]. Cells were used between passages 14 and 25; all experiments were performed at least 3 times and cells were free of viruses and mycoplasma. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 15 mM Hepes, and 2.5 ng/ml hFGF2. Cells were triggered for differentiation on the second day post-confluence (designated as day 0) in DMEM/Ham's F12 media supplemented with 10 μg/ml transferrin, 10 nM insulin, 0.2 nM triiodothyronine, 1 μM dexamethasone, and 500 μM isobutyl-methylxanthine. Two days later, the medium was changed (dexamethasone and isobutyl-methylxanthine removed), and 100 nM rosiglitazone were added. At day 9, rosiglitazone was withdrawn to enable white adipocyte differentiation. Rosiglitazone (100 nM) or GW7647 (300 nM) was added at day 14 to promote white to brite adipocyte conversion and cells were used at day 18.

Transfection experiments were performed using HiPerfect (QIAGEN, France) at day 14 of differentiation. Cells were incubated with a mixture containing HiPerfect and siRNA (50 nM) in DMEM. Four hours later, the mixture was supplemented with F12 medium containing 20 μg/ml transferrin, 20 nM insulin, and 0.4 nM triiodothyronine. siRNA against human DRP1 was purchased from Ambion (Life Technologies, Courtaboeuf, France) and validated to specifically target DRP1 (ID #: s19560).