Fig. 4. Damaged mitochondria are secreted via a pathway distinct from small EVs.
a Conditioned media from WT and ATG7-KO HeLa stably expressing mCherry-Parkin treated with A/O for 24 h were collected. Extracellular vesicles (EVs) were isolated by differential ultracentrifugation and immunoblotted for mitochondria and small EV markers. b Control or muscle specific ATG7-KO (Atg7f/f;Ckmm-cre) mice were subjected to 3 consecutive days of exhaustive exercise. Serum EVs or TA muscle tissue were then harvested. Representative immunoblots and quantification of the mitochondria markers (ACO2 and SDHA) from serum EVs are shown. Mean of n = 4 mice per group ±SEM. c Nanoparticle tracking analysis of EVs from WT and ATG7-KO cells untreated or treated with A/O for 24 h. d Conditioned media from 24 h A/O treated ATG7-KO HeLa stably expressing mCherry-Parkin were filtered (0.22 µm cutoff) before isolation of EVs by differential ultracentrifugation and immunoblotting. e Proteinase K protection assay of EVs isolated by differential ultracentrifugation from 24 h A/O treated ATG7-KO HeLa stably expressing mCherry-Parkin. f EVs from 24 h A/O treated ATG7-KO cells were separated by a 5–40% bottom-up iodixanol density flotation gradient and subjected to immunoblotting. *Non-specific bands.