Enzyme complex I (NADH-ubiquinone reductase), complex IV (cytochrome c oxidase, CcO) activity, and ATP levels were determined as described previously [7 (link)]. Briefly, cybrid cells were washed with ice-cold PBS, and then harvested, centrifuged, and suspended in 50 l of isolation buffer containing 250 mM sucrose, 20 mM HEPES, and 1 mM EDTA. Cell suspensions (containing ~3–4 mg of protein/ml) were added to a cuvette containing 0.95 ml of 1 × assay buffer (10 mM Tris-HCl, and 120 mM KCl), and the reaction volume was brought to 1.05 ml with the addition of 1× enzyme dilution buffer (10 mM Tris-HCl, pH 7.0). The reaction was then initiated by the addition of 50 μl of ferrocytochrome substrate solution (0.22 mM). The change in absorbance of cytochrome c at 550 nm was measured using a Shimadzu (Kyoto, Japan) UV1200 spectrophotometer. Activity is expressed as micromoles of cytochrome oxidized per min−1 mg−1 protein using an extinction coefficient of 18.64 mM−1 cm−1. ATP levels were determined using an ATP Bioluminescence Assay Kit (Roche) following the manufacturer’s instruction [9 (link), 25 (link)]. Briefly, cells were harvested, incubated on ice for 15 min, and centrifuged at 13,000 g for 10 min. ATP levels were measured using a Luminescence plate reader (Molecular Devices) with an integration time of 10 s.