ECAR and oxygen consumption rate (OCR)

The ECAR, OCR, and % metabolite dependency was measured using the Seahorse XFp Extracellular Flux Analyzer (Seahorse Bioscience). All experiments were performed according to the manufacturer’s protocol. Seahorse XFp Glycolysis Stress Test Kit, Seahorse XFp Cell Mito Stress Test Kit, and Seahorse XFp Mito Fuel Flex Test Kit (Agilent Technologies) were used to measure ECAR, OCR, and % dependency respectively. Briefly, cells were harvested, and 30,000 cells were plated per well and cultured in a Seahorse XFp cell culture microplate with complete growth media for 10–12 hr. The next day the cells were washed with Seahorse XF DMEM media (pH 7.4). The plate with cultured cells was placed in the Seahorse XFp Extracellular Flux Analyzer, and baseline measurements were recorded. For ECAR measurements, oligomycin, and 2-DG were sequentially injected into each well at the indicated time points. Similarly, for OCR measurements, oligomycin, FCCP (p-trifluoromethoxy carbonyl cyanide phenylhydrazone), and antimycin A (Rote/AA) were sequentially injected. For % dependency measurements, UK5099, BPTES, and Etomoxir were sequentially injected. The results obtained for ECAR, OCR, and Mito Fuel Flex Test were normalized to cell number. Data were analyzed using Seahorse XFp Wave software. The results for OCR are reported in pmols/minute, ECAR in mpH/minute, and Mito Fuel Flex Test in % dependency.