Supplementary Figure 10: An anti-HER2 EVIR encourages EV sorting to recycling endosomes.

(a) Representative confocal images of DC-EVIR (left) and DC-CtrlR (right) incubated with EV-HER2/mCh (magenta) for 1 or 5 h and immunostained for the indicated endocytic markers (green). The cells were stained with anti-dLNGFR antibodies (not shown) and their nuclei labeled with DAPI (blue). Scale bar, 10 μm. Images are representative of 10 cells randomly selected per condition.

(b) Co-localization analysis of mCh and the indicated endocytosis marker, measured using the Manders' coefficient. Data show mean values ± SEM (n=10 cells randomly selected per condition). Results derived from individual cells, rather than by averaging different cell culture replicates, are reported in order to illustrate the variability of the parameter of interest (degree of co-localization). Statistical analysis by unpaired two-sided Student's t test. Results are representative of 2 independent experiments.

(c) Ratio of EV recycling (in RAB11⁺ endosomes) over degradation (in LAMP1⁺ lysosomes), calculated from the data in (b). Statistical analysis as in (b). Results are representative of 2 independent experiments.(d) Schematic representation of endocytic pathways relevant to the data shown in panels (a-c).

Numerical values for the experiments with quantitative data are presented in Supplementary Table 2.