Fig. 7. Parkin activation inhibits the inclusion of mitochondrial proteins within EVs.

a AA treatment induces MDV formation. WT MEFs grown on coverslips were incubated in the absence (blue) or presence of AA for 1 h (red). mtHSP70 and TOM20 MDVs were then quantified. Each data point represents one cell. Bars represent the average of at least 30 cells in a minimum of three independent experiments ± SD. Two-sided t-test. b, c Damage-induced MDVs are targeted to lysosomes. b WT MEFs were treated as in (a) in the presence of E64/Pepstatin and MDV associaton with the lysosomal marker LAMP1 quantified as the fraction of total MDVs associated with LAMP1. Alternatively (c) LAMP1-positive MDVs were quantified from OPA1 KO MEFs. Each data point represents one cell. Bars represent the average of at least 20 cells in a minimum of three independent experiments ± SD. Two-sided t-test). d Stable expression of GFP-Parkin increase MDV formation. U2OS cells stably expressing GFP (blue) or GFP-Parkin (red) were treated with AA for 4 h and MDVs quantified. Each data point represents one cell. Bars represent the average of at least 20 cells in a minimum of three independent experiments ±  SD one-way ANOVA. e mtHSP70 MDVs association with Parkin. U2OS cells stably transfected with GFP-Parkin were treated as in (d) and the association of mtHSP70 MDVs quantified by immunofluorescence. Individual points represent independent experiments (n = 4). Bars show the average percent of the total (blue) and Parkin-positive (green) mtHSP70 MDVs ± SD. Two-sided t-test. f GFP-Parkin expression impairs the inclusion of IM/matrix proteins into EVs. EVs were isolated from U2OS cells stably expressing GFP (blue) or GFP-Parkin (red) and the indicated mitochondrial proteins measured as in Fig. 1. Individual points represent independent experiments (n = 3, except for TOM20 where n = 4). Bars show the average ±SD. Two-sided t-test.