Panel A contains a schematic illustration of reduced and oxidized Hyper7, fluorescence microscopy images of import-competent and import-defective mitochondria showing reduced and oxidized signals, merged images, and a line graph of normalized fluorescence versus distance from the cell centre in micrometers. Panel B contains violin plots showing the ratio of reduced to oxidized su9-Hyper7 versus normalized distance from the cell centre, comparing mitochondrial populations and treatment conditions, with statistical significance values indicated. Panel C shows overlaid density histograms of mitoSOX intensity for mitoQ-treated, NACA-treated, and untreated samples. Panel D presents fluorescence microscopy images of import-competent and import-defective mitochondria following NACA treatment, including individual and merged views. Panel E presents fluorescence microscopy images of import-competent and import-defective mitochondria following mitoQ treatment, including individual and merged views. Panel F contains scatter plots showing mean size of transferred mitochondria per cell in square micrometers and number of transferred mitochondria per cell under NACA and mitoQ treatment conditions.
ROS production dictates the fate of incoming mitochondria . (Ai) Schematic of Hyper7 oxidation by hydrogen peroxide. (Aii) Representative image of su9-Hyper7-expressing cell line (top panel, import-competent mitochondria) and su9-Hyper7;su9-mScarlet-DHFR-expressing cell line in the presence of 100 nM MTX (bottom panel, import-defective mitochondria). (Aiii) normalized fluorescence intensities taken along the red line marked on import-competent cell in Aii of emission band pass (BP) 525/50 in response to excitation at 405 nm (reduced Hyper7) or excitation at 488 nm (oxidized Hyper7) vs. distance from cell center. (Bi) reduced/oxidized su9-Hyper7 ratio in HeLaGAL cell line vs. normalized distance from cell center. n = 17 cells over N = 3 technical repeats. Mean lines shown on the graph. (Bii) reduced/oxidized su9-Hyper7 ratio in HeLaGAL cell line (import-competent mitochondria) in comparison with HeLaGAL cell line transfected with su9-mScarlet-DHFR and treated with 100 nM MTX for 48 h (import-defective mitochondria) vs. normalized distance from cell center. Error bars represent standard deviation. n = 17–19 cells over N = 3 technical repeats. P values from one-way ANOVA, multiple comparisons corrected for by the Dunnett test. Data distribution was assumed to be normal, but this was not formally tested. (C) mitoSOX intensity of HeLaGAL cells in response to 48 h treatment with 750 μM NACA or 100 nM mitoQ in comparison with no treatment. (D) Representative images of double-positive cells from a 48 h co-culture of su9-mTagBFP2 and su9-mScarlet-DHFR cell lines in the presence of MTX and NACA. (E) Representative images of double-positive cells from a 48 h co-culture of su9-mTagBFP2 and su9-mScarlet-DHFR cell lines in the presence of MTX and mitoQ. (F) Quantification of (i) mean size of transferred mitochondria per cell with NACA addition; (ii) number of transferred mitochondria per cell with NACA addition; (iii) mean size of transferred mitochondria per cell with mitoQ addition; (iv) number of transferred mitochondria per cell with mitoQ addition. n = 40 cells from N = 3 technical replicates. Mean lines shown on the graph. Scale bars 20 μm. All quantification was done on max. projected images.