Supplementary Figure 9: An anti-HER2 EVIR promotes EV internalization preferentially through macropinocytosis.

(a) The left panels show representative confocal images of DC-CtrlR and DC-EVIR after incubation with PKH67-labeled EV-HER2 (green) for 2 h, followed by washing with trypsin and acetate (or PBS), prior to fixation. The cells were stained with anti-dLNGFR antibodies (white) and their nuclei labeled with DAPI (blue). Scale bar, 10 μm. The right panel shows the quantification of the PKH67⁺ area; the total cell area was determined by dLNGFR⁺ membrane staining. Data show mean percentage values ± SEM (n=10 dLNGFR⁺ 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 (PKH67⁺ area). Statistical analysis by two-way ANOVA with Sidak's multiple comparison test. Results are representative of 2 independent experiments.

(b) The left panels show representative confocal images of DC-CtrlR and DC-EVIR treated with the indicated endocytosis inhibitors, followed by incubation with PKH67-labeled EV-HER2 (green) for 2 h, prior to fixation. The cells were stained with anti-dLNGFR antibodies (not shown) and their nuclei labeled with DAPI (blue). Scale bar, 10 μm. The right panel shows the analysis of the PKH67⁺ area, calculated as in (a). Data show mean percentage values ± SEM (n=10 dLNGFR⁺ cells randomly selected per condition). For statistical analysis, see (a). In addition, one-way ANOVA analysis with Dunnett's multiple comparison test was performed, within either DC-CtrlR or DC-EVIR group, to compare each treatment condition with the corresponding untreated group. Results are representative of 2 independent experiments.

(c) The left panels show representative confocal images of DC-CtrlR and DC-EVIR exposed to PKH67-labelled EVs (green) along with dextran-TRITC (magenta) or transferrin-AF555 (magenta) for 2h, prior to fixation. The cells were stained with anti-dLNGFR antibodies (not shown) and their nuclei labeled with DAPI (blue). Scale bar, 10 μm. The right panel shows co-localization analysis of mCh with the indicated endocytosis marker, measured using the Manders' coefficient. Data show mean values ± SEM (n=10 dLNGFR⁺ cells randomly selected per condition). For statistical analysis, see (a). Results are representative of 2 independent experiments.

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