Extended Data Fig. 4: Defective mitochondria are preferentially extruded by evading MYO6-mediated retention.
From: An extracellular vesicle-mediated mitochondrial transfer network critical for testosterone synthesis
a, Representative western blot images of MA-10 cells stably transfected with either a non-targeting scrambled shRNA (sh-Scr) or an shRNA targeting the Kif5b gene (sh-Kif5b) (n = 3 biological replicates per group). b, Quantification of KIF5B protein levels in MA-10 from the two groups (n = 3 biological replicates per group). c, Representative western blot images of MA-10 cells stably transfected with either a non-targeting scrambled shRNA (sh-Scr) or an shRNA targeting the Myo6 gene (sh-Myo6) (n = 3 biological replicates per group). d, Quantification of MYO6 protein levels in MA-10 from the two groups (n = 3 biological replicates per group). e, Representative immunofluorescence images of the MA-10 cells from different groups (n = 4 biological replicates per group). Scale bars, 15 μm. Inset shows boxed regions that are magnified. Scale bars, 5 μm. f, Flow cytometry analysis of the proportion of mitoDsRed+ particles among the GFP+ particles secreted by MA-10 in sh-Scr, sh-Kif5b or sh-Myo6 groups (n = 4 biological replicates per group). g, Quantification of mitoDsRed+ GFP+ particles by flow cytometry (n = 4 biological replicates per group). h, The gating strategy for MA-10 mitochondria with low TMRE (the lower 20%) and high TMRE (the higher 20%) via FACS after hCG (10 IU/L) treatment. TMRE was used to stain for MMP. mito, TMRE+ mitochondria; L, low TMRE; H, high TMRE (n = 3 biological replicates). i, Detection and quantification of the mean fluorescence intensity (MFI) of KIF5B–AF488 in low TMRE or high TMRE mitochondria (n = 3 biological replicates per group). j, Detection and quantification of the MFI of MYO6–AF488 in the two groups by flow cytometry (n = 3 biological replicates per group). k, Representative confocal images of the testes from Cyp17a1Cre; R26tdTomato mice. The testes sections were counterstained with F4/80 and early endosome marker (RAB5) (n = 3 mice). Scale bars, 2 μm. l, Representative confocal images of the testes from Cyp17a1Cre; R26tdTomato mice. The testes sections were counterstained with F4/80 and late endosome marker (RAB7) (n = 3 mice). Scale bars, 2 μm. Data are represented by mean ± SEM (b,d,g,i,j). Significances were determined using one-way ANOVA (g) or Two-tailed Student’s t-test (b,d,i,j). Source numerical data and unprocessed blots are provided.
