ATP synthesis assay

ATP synthesis assays were performed essentially as described (Bird et al., 2014 ). Briefly, 10 μg of cultured HEK293 cells in technical duplicates were permeabilized with 50 μg/ml digitonin in ATP assay buffer (25 mM Tris, 150 mM KCl, 2 mM EDTA, 10 mM K₂HPO₄, pH 7.4) containing 1 mM ADP and the indicated substrate/inhibitor concentrations (succinate [1 mM], glutamate [10 mM], malate [10 mM], pyruvate [10 mM], rotenone [2.5 μM], malonate [1 mM]). Samples were incubated at 37 °C for 20 min and then transferred to ice. Reactions were stopped with the addition of 0.6 M perchloric acid and neutralized using a 2 M KOH/0.6 M MOPS solution. The ATP concentrations in each reaction were measured in a microplate reader (BMG Labtech, FLUOstar Omega) using the ATP Bioluminescence Assay Kit CLS II (Roche, 11699695001). ATP synthesis rates (nmol/mg protein/min) of each samples were averaged from four biological replicates (n = 4).