Mitochondria isolation

For mitochondria isolation, all procedures were performed on ice or at 4°C. U2OS cells were grown to confluency in 150 mm Petri dishes and washed three times with 15 mL of cold homogenization buffer (10 mM HEPES, 1 mM EDTA, 210 mM mannitol, 70 mM sucrose, pH 7.4 at 4°C). Cells were harvested by scraping in cold homogenization buffer (0.75 mL per plate) supplemented with 1× protease inhibitor cocktail (MilliporeSigma, Burlington, MA) and lysed with 6–8 strokes of a glass Dounce homogenizer fitted with a tight pestle. At this point, a small fraction of homogenate was immediately snap-frozen on liquid nitrogen and stored at −80°C for whole-cell proteomics analysis as described below. For K562 suspension cells, cells were harvested by centrifugation (1000 g, 5 min), washed with cold homogenization buffer, re-pelleted (1000 g, 5 min), and incubated on ice for 20 min in swelling buffer (10 mM HEPES, 1 mM EDTA, pH 7.4 at 4°C) supplemented with 1× protease inhibitor cocktail (MilliporeSigma, Burlington, MA). Cells were then lysed with 25 strokes of a glass Dounce homogenizer fitted with a tight pestle and immediately diluted with 2× homogenization buffer (10 mM HEPES, 1 mM EDTA, 420 mM mannitol, 140 mM sucrose, supplemented with 1× protease inhibitor cocktail, pH 7.4 at 4°C) to a final concentration of 10 mM HEPES, 1 mM EDTA, 210 mM mannitol, 70 mM sucrose. The homogenate was centrifuged at ~1300 g for 5 min to remove nuclei, unbroken cells, and large cellular debris and the supernatant was centrifuged at ~14,000 g for 10 min at 4°C. The crude mitochondrial pellet was resuspended in homogenization buffer supplemented with 1× protease inhibitor cocktail prior to measuring protein concentration using a bicinchoninic acid (BCA) assay (Pierce, Waltham, MA). Mitochondrial samples were either used immediately or snap-frozen in 50 or 200 µg aliquots on liquid nitrogen and stored at −80°C.