Adipocyte maintenance medium

Human subcutaneous pre-adipocytes were purchased from Zen-Bio Inc. Specific lot numbers were selected based on BMI, gender, and age. Information on the donors is available in the Supplementary Information as Supplementary Table S1. Cells were cultured in Pre-adipocyte Medium (Zen-Bio Inc., Research Triangle Park, NC) at 37 °C, 5% CO₂. For differentiation to mature adipocytes, pre-adipocyte cells were plated at 18,000 cells/cm² and maintained in Pre-adipocyte Medium for 2–4 days until they reached 100% confluency (Day 0). Number of cells was counted using the Vision Cellometer^® (Nexcelom Bioscience, Lawrence, MA). Medium was replaced with Adipocyte Differentiation Medium (Zen-Bio Inc.) and cells were incubated for 7 days. Then, cells were fed with Adipocyte Maintenance Medium (Zen-Bio Inc.) every 2–3 days until complete differentiation was achieved at Day 14, according to the manufacturer’s instructions. Cultured mature adipocytes were washed 3 times with sterile DPBS and then starved overnight in serum- and antibiotic-free DMEM/Ham’s F12 medium (1:1) with l-glutamine and 15 mM HEPES (i.e. basal medium) before adding any treatments. For experiments with cycloheximide, differentiated cells were starved and pretreated with 0.178 mM cycloheximide diluted in DMSO 1 h prior to treatment with 0.05 µM insulin for 3 h.

PGE₂ was obtained from Cayman Chemicals (Ann Arbor, MI). Krebs-Ringer bicarbonate buffer, Dulbecco's Modified Eagle's Medium (DMEM), fatty acid-free (FAF)-BSA and liberase were from Roche (Basel, Switzerland). Nylon mesh filters (100 μm) were obtained from BD Biosciences (San Jose, CA). TRIzol was from Invitrogen (Carlsbad, CA) and L-Glutamine from Biological Industries (Kibbutz Beit Haemek, Israel). FBS and Dulbecco’s PBS with and without calcium and magnesium (DPBS^+/+ and DPBS^-/-, respectively) were obtained from Lonza (Vervieres, Belgium). The free glycerol assay kit was from Sigma (St. Louis, MO). The High-Capacity Archive Kit and TaqMan Gene Expression Assays were provided by Applied Biosystems (Foster City, CA). Adipocyte maintenance medium was from Zen Bio (Research Triangle Park, NC). The Pierce BCA Protein Assay kit was from Thermo Scientific (Carlsbad, CA).