Dmem f12

For adipocyte differentiation, ASC were amplified until 80% of confluency was reached and culture medium replaced by differentiation medium containing Dulbecco's modified Eagle medium/Nutrient Mixture F12 (DMEM/F12; Dominique Dutscher, France), FBS 10% (Hyclone, France), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 1 μg/ml insulin, 0.5 μM dexamethasone, 2 nM Triiodo-L-Thyronine 3 (T3), 0.5 μM 3-isobuthyl-1-methylxanthine (IBMX), 2 μM rosiglitazone, and 10 μg/ml transferrin, all from Sigma-Aldrich. After 7 days, adipocyte differentiation was measured by Oil red O staining of the neutral triglyceride and lipids. Cells were fixed in 10% formaldehyde (Carlo Erba, France) for 1 hour then washed with 60% isopropanol (Carlo Erba) twice, dried and colored by incubation for 10 minutes with 0.2% Oil red O (Sigma-Aldrich) in isopropanol. Brightfield images were taken with an optic microscope with a ×20 magnification. The expression of adipogenic genes (Pparγ, Dgat2, and Hsl) was measured by RT-qPCR.

Three human and murine melanic lines from the “American Type Culture Collection” (ATCC) were used: the SK-ML-28 line established from a primary skin tissue tumor from a Caucasian human, the MelC line derived from a grade IV human metastatic melanoma tumor, and the murine B16F10 line derived from lung metastases in mice (C57BL/6jRJ). The SK-ML-28 and MelC cell lines have an activating mutation of the BRAF serine/threonine kinase protein (BRAFV600E), while the B16F10 cells express the wild-type form of BRAF. These three cell lines do not possess mutations of the p53 protein and express the wild-type form of the protein. The cultured cells were maintained in Dulbecco’s Modified Eagle F12 medium (DMEM/F12) supplemented with 10% fetal bovine serum (Dutscher, Brumath, France) and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO₂ at 37 °C.

The human retinal pigmented epithelial cell line ARPE-19, purchased from the American Type Culture Collection (Manassas, VA, USA), were maintained in Dulbecco’s modified Eagle’s F12 medium (DMEM/F12) supplemented with 10% fetal bovine serum (Dutscher, Brumath, France), 1% penicillin/streptomycin in a humidified atmosphere of 5% CO₂ at 37 °C. Cells were seeded and grown to a subconfluence of 60–70% in normoxia. Twenty four hours after seeding, the medium was removed and the cells were washed once with Hank’s Balanced Salt Solution (HBSS, Dutscher, Brumath, France) before reincubating in DMEMF12 with 1% FBS and 1% penicillin/streptomycin. The following day, cells were treated with DMSO, RWE with indicated concentrations or resveratrol 20 µM.