Supplementary Figure 3: DCs transduced with an anti-HER2 EVIR capture and internalize HER2+ EVs.
From: EVIR: chimeric receptors that enhance dendritic cell cross-dressing with tumor antigens
(a) Representative flow cytometry dot plots of GFP and EVIR (stained with an anti-dLNGFR antibody) expression in DC-CtrlR and DC-EVIR cells. One cell culture per LV type is shown; data are representative of 3 independent cell cultures.
(b) Representative flow cytometry histograms of HER2 expression in EV-mCh and EV-HER2/mCh. One EV preparation per type is shown; data are representative of 2 independent EV preparations.
(c) Size distribution of EV-mCh and EV-HER2/mCh, determined by nanoparticle tracking analysis (NTA). Data show mean values ± SEM (n=3 technical measurements of one EV preparation per type). Data are representative of 3 independent EV preparations.
(d) Flow cytometry analysis of mCh in DC-EVIR either untreated or treated with EV-mCh or EV-HER2/mCh. Data are representative of 3 independent cell cultures per condition.
(e) Representative three-dimensional projections of confocal z-stack images showing nuclear DAPI (blue), anti-dLNGFR (green) and anti-mCh (magenta) immunostaining in DC-CtrlR and anti-HER2 DC-EVIR incubated for 2 h with MC38-HER2/mCh EVs. Scale bar, 10 μm. Images are representative of at least 30 cells per condition, analyzed from 3 independent experiments.
(f) Dose-response analysis of the median fluorescence intensity (MFI) of mCh in DC-CtrlR and DC-EVIR, either untreated or treated with EV-mCh or EV-HER2/mCh, and analyzed by flow cytometry. Data show mean values ± SEM (n=3 independent cell cultures per condition). Statistical analysis by two-way ANOVA with Sidak's multiple comparison test. The experiment was performed one time.
Numerical values for the experiments with quantitative data are presented in Supplementary Table 2.
