Fig. 4: Qualitative analysis of extracellular mitochondrial uptake.

A–D Confocal micrographs showing uptake of free mitochondria derived from Cherry-OMP25 expressing donor cells, internalized by acceptor cells for 4 h. After incubation, cells were fixed and processed for immunofluorescence against EEA1 A or LAMP1 C to label early endosomes and lysosomes, respectively, using green-labeled secondary antibodies. Red squares indicate regions of colocalization shown at higher magnification. B, D Quantification of colocalization. EEA1 or LAMP1 fluorescence was measured for each mitochondria-associated (red-positive) ROI, as well as for control EEA1-/LAMP1-negative ROIs. The dashed line indicates the maximum fluorescence intensity in EEA1-/LAMP1-negative ROIs. Values above this threshold were considered positive for colocalization. Three independent experiments were performed; 320 B and 324 ROIs D from at least 50 cells, from three independent samples were analyzed. Scale bar: 10 μm. E, F Confocal micrographs showing uptake of free mitochondria derived from Nluc-HA-OMP25–expressing donor cells after 4-h internalization. Acceptor cells were co-treated with Texas Red dextran and processed for immunofluorescence against HA using green-labeled secondary antibodies. Red squares highlight regions of colocalization at higher magnification. F Quantification of colocalization with dextran was performed as described above. Scale bar: 10 μm. 351 ROIs from 60 cells from three independent samples were analyzed.