Ebm 2 singlequots

The human hepatoma cell lines, HepG2, Huh7, Mahlavu and J7 were routinely grown in Dulbecco Modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). The human umbilical vein endothelial cells (HUVECs) line was growth in endothelial cell basal medium-2 (EBM-2; Lonza, CC-3156) supplemented with EBM-2 SingleQuots (Lonza, CC-4176). Stably transfected HepG2-TRα, HepG2-TRβ, and HepG2-neo cells were cultured in DMEM containing 10% (v/v) FBS with G418 23 (link). NUPR1-overexpressing (ovNUPR1) and luciferase control Mahlavu cells were cultured in DMEM containing 10% (v/v) FBS with blasticidin. NUPR1-knockdown (shNUPR1) and luciferase control (shLuc) Huh7 cells were cultured in DMEM containing 10% (v/v) FBS with puromycin. T₃ was purchased from Sigma-Aldrich (T2752). Serum was depleted of T₃ (0 nM T₃) using an AG 1-X8 resin (Bio-Rad, 40-1451). Cells were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO₂.

Endothelial cells were seeded at 20 000 cells/well on a Seahorse 96-well XF Cell Culture Microplate as detailed by the manufacturer (Seashore Bioscience/Agilent Technologies, Santa Clara, California, USA) and allowed to adhere overnight in complete EBM-2 (EBM-2 basal media plus EBM-2 SingleQuots, Lonza, Basel, Switzerland). The following day cells were rinsed with 1× PBS and 50% control or SLE patient serum was added to wells and allowed to incubate for 24 hours (six samples per group with five replicates per patient sample). The Seahorse XF Analyzer (Seashore Bioscience/Agilent Technologies) was used to determine basal oxygen consumption rate (OCR). Four basal rate measurements were followed by four measurement cycles following each injection (1 µM oligomycin, 1 µM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and 2 µM AA rotenone). Consumption rates were calculated as previously described.33 (link) The bioenergetic health index (BHI) was calculated using the following formula: BHI= (ATP-linked × Reserve Capcity)/(Proton Leak × Non-mitochondrial OCR).34