Figure 1.
Extracellular release of mitochondria. (A) Time-lapse confocal imaging on PC12DsRed2mito showed release of extracellular mitochondria from cells. (B) Extracellular mitochondria studied by CLEM. (i–ii) Merged image of fluorescence and brightfield was acquired to visualize the grid and locate the DsRed2mito-labeled particle (arrowhead) at EM level. (ii) Scanned area (arrowhead) became dark. (iii) Zoomed-in view of the scanned area showing most extracellular DsRed2mito-labeled particles are free mitochondria. (iv) Magnified view showing typical cristae and small matrix granules in a morphological intact mitochondrion (boxed area in Biii). (C)(I) Immuno EM showing some particles are TOMM20-positive (arrowhead) as indicated by 10 nm colloidal gold particles. (ii) Immuno-gold detection of mRFP (arrowhead, 6 nm colloidal gold) and TOMM20 (black arrow, 12 nm colloidal gold) showing double-positive labeling. (D) Western blot analysis confirming presence of TOMM20, HSPD1, OPA1, CI-NDUFB8, CII-SDHB, CIII-UQCRC2, CIV-MT-CO1, CV-ATP5F1A in CM-derived pellet. (E) Effect of apoptosis inhibitors on the TOMM20 level in CM-derived pellet. One-way ANOVA, Dunnett’s test, n = 3 for each group