Triiodothyronine t3

Antibodies to β-actin, tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and enhanced green fluorescent protein (eGFP) and Ahnak CRISPR/Cas9 plasmid were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Ahnak and Bmpr1α antibodies were obtained from Abcam (Cambridge, UK). Anti-Ppar-γ, perilipin-1 (PLIN1), phosphorylated Smad1/5 (pSmad1/5), Smad1, phosphorylated AKT (pAKT), AKT, phosphorylated extracellular signal-regulated kinase (ERK)1/2 (pERK1/2), ERK1/2 and C/EBPα antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). 3-Isobutyl-1-methylxanthine (IBMX) and triiodothyronine (T3) were obtained from Cayman Chemical (Ann Arbor, MI, USA), Dexamethasone (DEX), rosiglitazone (Rosi), insulin, and 0.5% Oil Red O were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum, Dulbecco’s phosphate buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM), and DMEM/F12 were purchased from WelGene (Daegu, Korea). Secrete-Pair Dual Luminescence Assay Kit and pEZX-Bmpr1α promoter plasmid were obtained from GeneCopoeia (Rockville, MD, USA)

Adipose tissues obtained from CIDEA reporter mice were processed for stromal vascular fraction isolation as previously described^{35 (link)}. For adipogenic differentiation, confluent cells were exposed to differentiation medium: growth medium (DMEM with 10% FBS and 1% penicillin–streptomycin) containing 2.5 mM of isobutylmethylxanthine (Cayman), 1 μM of dexamethasone (Cayman), 1 μg/ml of insulin (Sigma), 0.125 mM of indomethacin (Cayman), and 1 nM of triiodothyronine (T3, Cayman) for 3 days, and then exposed to maintenance medium: growth medium containing 1 μg/ml of insulin and 1 nM of T3 for 3 days. Differentiated adipocytes were treated with PMSFs (1 μM) in growth medium for 24 h. Zeiss LSM800 confocal microscope or Nikon Ts2-FL fluorescence microscope was used to detect fluorescence signals.