Blockade of nSMase-2 results in mitochondrial export deficits. (A) Average total number of unique proteins identified by quantitative LC-MS/MS of EVs derived from SMPD3-KO (EV^SMPD3-KO, n = 3) vs AAVS1-KO (EV^Con, n = 3) human CD34+ cells. (B) Principal component analysis (PCA) plot of vesicle proteomes. (C) Overlap in unique proteins identified in EV^SMPD3-KO and EV^Con. (D) Proportions and (E) volcano plot of depleted and enriched proteins differentially abundant in EV^SMPD3-KO compared with EV^Con. The dotted horizontal line represents −log₁₀(P = .05). (F) String analysis of protein-protein networks depleted in EV^SMPD3-KO. Gene ontology analysis of the (G) molecular function or (H) cell compartment of proteins significantly depleted in EV^SMPD3-KO. (I) Representative immunoblot analysis and (J) quantitation of mitochondrial proteins depleted in EVs derived from HSPC^GW, equal volume loaded. (K) Total DNA extracted from HSPC^GW vesicles after 2000g (2K), 10 000g (10K), or 100 000g (100K) centrifugations; the dotted line indicates average normalized EV DNA extracted from HSPC^Con vesicles. (L) Relative copy number of mtDNA regions per genes in cells and 100K vesicles after GW4869 treatment; dotted line represents normalized controls. Representative fluorescence-activated cell sorter plots (M) and quantification of MitoTracker staining in 100K vesicles (N). Relative chimerism measured over weeks after competitive transplantation of Tsg101-KO (n = 8) (O) or Cd63-KO (n = 7) (P) vs Rosa26-KO murine HSPCs (n = 15). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. Refer to supplemental Figures 5 and 6.