Fig. 3.

Mitochondrial components segregate into MVBs and are secreted in exosomes. a Confocal co-localization analysis in HEK293 cells treated with FCCP (4 h) and stained with SSBP1 (red), EEA1 and CD63 (green). Bar, 10 µm. Graph shows means for Mander’s co-localization coefficient for SSBP1 and EEA1 or CD63 (n = 3). t-test: ***P-value < 0.0001. b Confocal co-localization analysis in HEK293 cells co-transfected with Rab7-Q67L-GFP (Rab7mutGFP, green) and mitoDsRed (red), and immunostained for TFAM (purple) and nuclei (HOECHST 58, blue). Center and right images show high magnification of left images (Rab7-Q67L-GFP and mitoDsRed in upper panels; Rab7-Q67L-GFP and TFAM in lower panels). Charts: Fluorescence profiles along the corresponding white lines. Bar, 10 µm. c Mitochondrial components in the exosome fraction obtained from equal numbers of shControl and shnSMase2 J77 T cells. ATAD3, TFAM, and CD63 were detected by immunoblot; mtDNA was detected by PCR for HVRII. Graph: Quantification of exosomal proteins and mtDNA in a representative experiment (n = 3). d Flow cytometry analysis of mitochondrial mass (Mitotracker green), intracellular ROS levels (DCFDA staining), oxidized DNA (8-OHdG Ab staining), and endogenous TFAM in HEK293 cells knocked down for nSMase2 and Rab27a. Graphs: Quantification from 5–8 independent experiments (Mean). t-test *P-value < 0.05; **P-value < 0.001. e Electron microscopy images show defects in mitochondrial ultrastructure and cristae organization in shnSMase2 and shRab27a HEK293 cells. Graph: Quantification of mitochondrial cristae width (Mean). t-test: ***P-value < 0.0001. f Graph: Basal oxygen consumption rate (OCR) of control, shnSMase2 and shRab27a HEK293 cells. Dots represent mean from three independent experiments run in duplicate or triplicate. t-test, *P-value < 0.05, ***P-value < 0.0001. Chart: OCR from shControl, shnSMase2 and shRab27a HEK293 cells in response to oligomycin (Oligo), fccp, and rotenone plus antimycin A (Rot/AA). (n = 2; mean ± S.E.M.). g Western blot analysis of exosomes from Jurkat T cells left untreated, serum-starved overnight or treated with bafilomycin A. Membranes were blotted for TFAM, CD81, and CD63. Graph: Nanoparticle concentration in the exosomal fractions (mean, two independent experiments). h Western blot analysis of exosomes obtained from Jurkat T cells transfected with control or LC3 siRNA. Graph: Nanoparticle concentrations in the exosomal fractions (mean, n = 2). Western blots are representative out of three independent experiments