3 5 3 triiodothyronine t3

The Stromal vascular fractions (SVFs) of BAT were isolated as previously described12 (link) with modification. Freshly isolated BAT pads from male 6-week-old A-FABP KO mice and their relative WT littermates were minced and digested in 0.1% (w/v) collagenase type I (Invitrogen, CA, USA) for 30 min at 37 °C with gentle shaking in water bath. The digestion mixture was passed through a 70 μm cell strainer (BD Biosciences) and centrifuged at 700g for 5 min at 4 °C. The SVFs were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (Invitrogen, CA, USA) and 1% penicillin and streptomycin (PSF, Thermo Fisher Scientific, USA) until confluence. To differentiate SVFs into mature adipocytes, SVFs cells were treated with insulin (20 nM, Novartis, Swiss), 3-isobutyl-1-methylxanthine (0.5 mM, Sigma-Aldrich), dexamethasone (1 μM, Sigma-Aldrich), 3,3,5-triiodothyronine (T3, 1 nM, Sigma-Aldrich), indomethacin (125 nM, Sigma-Aldrich), retinoic acid (20 μM, Sigma-Aldrich) and Vitamin C (142 μM, Sigma-Aldrich) in DMEM for 2 days then changed to DMEM containing insulin (20 nM) and T3 (1 nM) for another 4 days.

Dulbecco’s modified Eagle’s medium (DMEM)-high glucose and collagenase were purchased from Wako Pure Chemicals (Osaka, Japan). Fatty acid-free bovine serum albumin (BSA), isobutylmethylxanthine (IBMX), dexamethasone (Dex), insulin, 3,3′,5-triiodothyronine (T3), and troglitazone (Tro) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal calf serum (FCS) was obtained from Trace Scientific Ltd. (Melbourne, Australia). Rabbit anti-serum against UCP1 was a gift from Dr. Teruo Kawada (Kyoto University, Japan). Antibodies against aP2, caspase-9, and the phosphorylated form of Rb were purchased from Cell Signaling Technology (Beverly, MA, USA); those against β-actin, p57, and a-Tubulin were from Sigma-Aldrich; those against p27 and Rb were from BD Biosciences (San Jose, CA, USA); that against PCNA was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and those against Ki67 and MCT1 were from Abcam (Cambridge, MA, USA). Anti-rabbit and anti-mouse secondary antibodies conjugated with horseradish peroxidase were obtained from Cell Signaling Technology. Anti-rabbit or anti-chicken secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 594 were obtained from Thermo Fisher Scientific (Gaithersburg, MD, USA).

Rg3, insulin, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), indomethacin, and 3,3′,5-triiodothyronine (T3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A lipid (Oil-Red O) staining kit was purchased from BioVision (Mountain View, CA, USA). 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was purchased from ThermoFisher Scientific (Waltham, MA, USA). A triglyceride (TG) assay kit was obtained from Abcam (Cambridge, UK). Antibodies against PPARγ, perilipin, endoplasmic reticulum oxidoreductin 1 alpha (ERO1L), phosphorylated eukaryotic initiation factor 2 alpha (Phospho-eIF2α), phosphorylated Akt (Phospho-Akt), total Akt, cleaved caspase-3, procaspase-3, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxide-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).