Mitochondrial Bioenergetics and Oxidative Stress Assays

OCR and ECAR values were determined using the XF24 Extracellular Flux Analyzer (Seahorse Bioscience) following the manufacturers’ protocols. For β-oxidation, WT MEFs and shRNA-mitoN MEFs (60,000 per well; 1 µg ml⁻¹ Dox) were seeded overnight in a XF24 cell-culture microplate at 37 °C under 5% CO₂ (Seahorse Bioscience). Following 1 hr equilibration with 1X KHB buffer (111 mM NaCl, 4.7 mM KCl, 2 mM MgSO₄, 1.2 mM Na₂HPO₄) supplemented with 0.5 mM carnitine and 2.5 mM glucose, cells were subjected to treatment of a palmitate-BSA-conjugate (200 µM; C:16:0), followed by etomoxir (100 µM). OCR measurements were recorded at set interval time-points. For electron-flow (EF) measurements, isolated mitochondria were pelleted in a XF24 cell-culture microplate by centrifugation (2,000 g for 20 min at 4 °C) in 1X MAS buffer (70 mM sucrose, 220 mM mannitol, 10 mM KH₂PO₄, 5 mM MgCl₂, 2 mM HEPES, 1 mM EDTA in 0.2% FA-free BSA) supplemented with 10 mM pyruvate, 10 mM malate and 4 µM FCCP. OCR and ECAR measurements were obtained following sequential additions of rotenone (2 µM final concentration), succinate (10 mM), antimycin A (4 µM) and ascorbate (10 mM) (the latter containing 1 mM TMPD). For ECAR glycolytic flux experiments using whole-tissue slices, oligomycin (2 µM), FCCP (4 µM), 2-DG (100 mM) and antimycin A (10 µM) were added to tissues in an XF24 islet-capture Microplate (Seahorse Bioscience). All compounds and materials above were obtained from Sigma-Aldrich. For mitochondrial oxidative-stress, the protein-carbonylation assay was performed as previously described^{37 (link)}. For lipid-peroxidation, bound 8-isoPGF2α levels were determined as previously detailed^{19 (link)}. The mitochondrial membrane potential (ΔΨm) experiments are detailed in the Supplementary Methods.