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Figure S3. Analyses of mitochondrial contiguity in cells expressing high levels of MARCH5^H43W. Interconnectivity of mitochondria was analyzed in MARCH5^H43W-CFP and mito-YFP expressing cells with apparent rounding and perinuclear accumulation of these organelles. The round mitochondrion (region of interest; ROI 1; A) was bleached, followed by imaging of fluorescence recovery (B) that is predicted to occur if the bleached area is connected with the bulk mitochondrial network. Photobleach (indicated with “V”) and recovery cycles were repeated seven times. Images 1-5 correspond to the time points indicated on the recovery curves. Note efficient recovery of fluorescence in photobleached ROI 1 (B) over the time of experiment, and delayed response to photobleaching of the mitochondrion (ROI 2; C) adjacent to ROI 1 indicating an exchange of matrix content between these two mitochondria. Negligible changes in fluorescence intensity of ROI 3 (D) indicate low nonspecific photobleaching and validate fluorescence changes occurring in ROIs 1 and 2.