Seahorse assay.

The Seahorse assay was performed using the Agilent Seahorse XFe24 extracellular flux assay kit (Agilent Technologies) according to the manufacturer’s protocol. Briefly, ERRα was knocked down in HUVEC using siRNA. Thirty hours posttransfection, cells were plated in the assay plate at a density of 40,000 cells/well. The assay was performed 48 to 60 h posttransfection. Cells were incubated in the assay medium containing 25 mM glucose, 2 mM glutamine, and 2 mM sodium pyruvate at 37°C for 1 h prior to measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). After measurement of baseline OCR and ECAR, cells were treated sequentially with oligomycin (1 μM), FCCP (2 μM), and antimycin A-rotenone (0.5 μM), followed by measurement of OCR and ECAR after every measurement. Fatty acid oxidation was measured using the XF palmitate-BSA FAO substrate (Agilent Technologies) by adhering to the manufacturer’s protocol. Cells were incubated with 150 μM palmitate conjugated to 250 μM BSA. OCR and ECAR were measured at baseline, followed by measurement after each sequential drug treatment of oligomycin (2 μM), FCCP (3 μM), and antimycin A (2 μM)-rotenone (4 μM).