Supplementary Figure 4: DCs transduced with an anti-HER2 EVIR show accelerated kinetics of EV and mCh internalization.

(a) Time course analysis of mCh localization in DC-EVIR or DC-CtrlR cells incubated with EV-HER2/mCh. The top panel shows representative confocal images of randomly selected dLNGFR⁺ cells, analyzed at the indicated time points after exposure to EVs. Data show anti-dLNGFR (green) and anti-mCh (magenta) immunostaining, and nuclear staining with DAPI (blue). Scale bar, 10 μm. The bottom panel shows analysis and quantification of the subcellular localization of the mCh signal in DC-CtrlR and DC-EVIR cells. Data show percentage values ± SEM (n=8 dLNGFR⁺ cells randomly selected per time point and 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 (mCh localization in 128 independent cells from one experiment). Results are representative of 2 independent experiments. UT, untreated cells (not incubated with EVs).

(b) Flow cytometry analysis of mCh or mTq fluorescence in iBMM-EVIR cells, either untreated or treated with the indicated EV preparations. Data illustrate the MFI of mCh (top) and mTq (bottom), shown as mean values ± SEM (n=3 independent cell cultures per condition). Statistical analysis by one-way ANOVA with Tukey's multiple comparison test.

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