O4876

XF24 extracellular flux analyzer (Seahorse Bioscience) was used to measure the oxygen consumption rate following the manufacturer’s instructions. Briefly, an XF24 cartridge was equilibrated with the XF calibrant overnight at 37°C. DMEM was used to prepare reagents and serve as the assay buffer. The compounds used for mitochondrial respiration assay were oligomycin (0.5 μM, O4876, Sigma), carbonyl cyanide-4-phenylhydrazone (FCCP) (2.5 μM, C2920, Sigma), and rotenone (0.5 μM). COUP-TFII-overexpressing cells were used in this experiment. Data were pooled from repeated independent experiments (5 replicates/each).

Seahorse analysis was performed on an XFe96 Analyser from Agilent Technologies (Santa Clara, CA, USA). Cells were plated at a density of 2.0 × 10⁴ cells per well for each cell line 24 h prior to analysis. Cells were washed twice with unbuffered Seahorse medium (Agilent #102353-100 DMEM, Santa Clara, CA, USA, with or without 3 mM L-glutamine and with 10 mM glucose) prior to adding Seahorse medium containing drugs. Cells were treated with the drugs for 10 min prior to starting the assay, during which the drugs remained present. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured during subsequent injections of 2.5 µM oligomycin (ATP synthase inhibitor, Sigma #O4876, St. Louis, MO, USA), 50 µM 2,4-dinitrophenol (DNP; uncoupler, Sigma #D19850, St. Louis, MO, USA), 2 µM rotenone with 4 µM Antimycin A (complex I and III inhibitors, respectively, Sigma #R8875 and #A8674, St. Louis, MO, USA) and lastly, 100 mM 2-deoxyglucose (2-DG; glucose analog and hexokinase II inhibitor, Sigma #D8375, St. Louis, MO, USA); these are final concentrations in the wells. The OCR:ECAR ratio was calculated from basal OCR and ECAR corrected for background levels of OCR and ECAR after 2-DG injection. Spare capacity was calculated by subtracting the basal OCR from the maximal OCR.