Mitophagy detection

Mitophagy was observed via immunofluorescence by co-staining of mitochondria and lysosome. The mitophagy flux was measured by detecting GFP-LC3II puncta through transfection of recombinant adenovirus GFP-LC3 under laser scanning confocal microscopy (Nikon A1R, Japan). Mt-Keima is a ratiometric pH-sensitive fluorescent protein that is targeted into the mitochondrial matrix. A low-ratio mt-Keima derived fluorescence (543/458 nm) reports neutral environment, whereas a high-ratio fluorescence reports acidic pH. Thus, mt-Keima enables differential imaging of mitochondria in the cytoplasm and mitochondria in acidic lysosomes. Mitochondria-targeted mKeima-Red expression plasmid (pMT-mKeima-Red, #AM-V-251, MBL Medical & Biological Laboratories, Co., ltd. Woburn, MA) was transfected to cells. Ratio (543/458 nm) of mKeima emission light were calculated as a value of mitophagy. Details on ROS measurement, ATP production, and mitochondrial respiratory function are described in the supplemental methods.