Fig. 1: Inhibiting lysosomal acidification leads to increased release of large and small extracellular vesicles (EVs) from cells and hearts.
From: Mitochondria are secreted in extracellular vesicles when lysosomal function is impaired
a Representative Western blots of whole cell lysate (WCL) and extracellular vesicle fractions for EV marker proteins Alix, Tsg101, CD81 and the mitochondrial proteins Tim23 and Tom20. The EVs were obtained by differential centrifugation of conditioned media from mouse embryonic fibroblasts collected over 48 h after treatment with vehicle or Bafilomycin (Baf A1, 5 nM). b Quantification of proteins in large EVs (Alix n = 8; Tsg101, Tim23 n = 7; Tom20 n = 5; CD81 n = 4 biologically independent experiments). c Venn diagram showing overlap of proteins detected in large and small EVs. d Gene ontology (GO) analysis of the unique 519 proteins identified in large EV fractions with the top 15 terms for cellular component plotted according to –log10False Discovery Rate (FDR). e Representative Western blot analysis of EV proteins CD63, CD81 and the mitochondrial protein Tim23 in heart lysates and EV fractions isolated from heart tissue. f Quantification of proteins in large EVs fraction (CD63 n = 5; DC81, Tim23 n = 4 biologically independent experiments). g Photo-activatable mitochondria (PhAMfloxed) mice were crossed with Myh6-Cre transgenic mice to generate a mouse line (PhAMexcised) with cardiac specific expression of Mito-Dendra2. Expression of Mito-Dendra2 (green) and Cre was confirmed by imaging of frozen heart sections (representative of n = 2 biologically independent samples) and by Western blotting of heart lysates (n = 3 biologically independent samples). Heart sections were stained with anti-Cytochrome c (red) to label mitochondria. Scale bar = 20 μm. h Representative Western blot analysis of CD63, CD81, Tim23, and Mito-Dendra2 in heart lysates and EV fractions isolated from heart tissue. i Quantification of proteins in large EVs (n = 5 biologically independent experiments). Data are mean ± SEM. ns = not significant. P values shown are by two-sided Student’s t-test. Source data are provided as a Source Data file.
