Rotenone antimycin a

Oxygen consumption rates (OCR) were measured using a Seahorse XFe96 analyzer (Agilent) where cells were seeded into Seahorse XFe96 plates at 10,000 – 20,000 per well overnight depending on the cell line. Respiration in intact cells was measured in DMEM (Sigma #5030) supplemented with 10 mM glucose and 2 mM glutamine. Where indicated, cells were pre-treated with 2 μM Antimycin A 15 minutes prior to initial measurements. Respiratory rates were measured in response to sequential injections of oligomycin (2 μM), FCCP (0.5 μM) and rotenone / antimycin A (1 μM) (all Cayman Chemicals). Permeabilized cell respiratory rates were measured after cells were washed and resuspended in 1x MAS buffer containing either substrates pyruvate (10 mM) and malate (1 mM) or ascorbate and TMPD, along with ADP (4 mM) and XF PMP Reagent (1 nM) (Agilent Technologies). Phosphorylating (“State 3”) respiration was measured as described in Divakaruni et al. (2014) . Cells were immediately lysed in 25 μL/well Reagent A (Bio-Rad) after each assay, and protein quantified using the DC protein assay kit (Bio-Rad). OCR was normalized to micrograms total protein, as quantified by a standard curve.