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Cells were then washed twice with mitochondrial assay solution (220 mM mannitol [Sigma-Aldrich, M4125], 70 mM sucrose [Sigma-Aldrich, S7903], 10 mM KH 2 PO 4 [VWR, 470302], 2 mM HEPES [Fisher Scientific, BP310], and 1 mM EGTA [VWR, 97062]) and then overlaid with 175 L of mitochondrial assay solution supplemented with 10 mM sodium pyruvate (Sigma-Aldrich, P5280), 1 mM malate (Sigma-Aldrich, M0875), 4 mM ADP (Sigma-Aldrich, A5285), and Seahorse Plasma Membrane Permeabilizer (Agilent, 102504-100)

This degradation occurs through multiple processes, including macroautophagy (enclosing large cellular components in autophagosomes for lysosomal delivery), microautophagy (engulfing and digesting segments of their own cytoplasm) and chaperone-mediated autophagy (CMA) (direct translocation of specific proteins to lysosomes) (3,4)

All standards were obtained from Cambridge Isotope Laboratory unless stated otherwise