Subcellular fractionation

For mitochondria preparation, the Mitochondria Isolation Kit (ab110171; Abcam) was used, according to the manufacturer’s instructions. Briefly, trypsinized cells were collected and pelleted by centrifugation at 1000g. After freezing and thawing several times, cells were resuspended in Buffer A and incubated for 10 min on ice, followed by dounce homogenization. After centrifugation, the supernatant was saved and the pellet was resuspended in Buffer B to the same volume as Buffer A, following the same rupturing steps. Combined supernatants were centrifuged at 12,000g for 15 min at 4°C to obtain the mitochondrial fraction. For the membrane fraction, after obtaining the mitochondrial pellet, the supernatant was collected and centrifuged at 100,000g for 1 hour in an ultracentrifuge. The pellet was resuspended in wash buffer and recentrifuged for 45 min. The membrane pellet was then resuspended in lysis buffer [250 mM sucrose, 20 mM Hepes (pH 7.4), 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 0.1% SDS, 1 mM dithiothreitol, and protease inhibitor (PI) cocktail]. Western blotting was used to detect the purity of the fractions with Tom20 as a mitochondrial marker, actin as a cytosolic marker, and PDI as a marker for ER.