Fig. 2

Caveolin-mediated endocytosis is implicated in EEV uptake by BMEC-like cells. (A-B) Representative confocal images of control BMEC-like cells stained for phalloidin (white) and DAPI nuclear stain, showing internalized EEVs (CellMask orange, white arrows) (A), and corresponding quantification of the number of internalized EEVs per BMEC-like cell (B) when the cultures were exposed to EEVs in the presence or absence of 5 µM Filipin or 6 mUI/mL heparinase III. Scale bars: 20 μm. (C-D) Nanoparticle FACS-based quantification of EEV transcytosis (C) and measure of monolayer integrity (D) in the transwell model when the BMEC-like cell growth medium located in the top chamber is supplemented with control EEVs, EEVs + 5 µM Filipin or vehicle control, absence of EEVs, or absence of BMEC-like cells (a positive control for 3 kDa dextran-TMRE penetration into the bottom compartment). Statistical analysis: Error bars represent mean + SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test (*p-value < 0.05; **p-value < 0.01) (B), or Wilcoxon signed rank test with theoretical median (untreated) set at 1 (*p-value < 0.05) (C-D). In this figure, a biological replicate consists of BMEC-like cells treated with EEVs that were produced from two different healthy donors (age = 30 years old) and data is averaged to generate a single value. Data points show three biological replicates collected using three different iPSC lines (line IDs 38554, 41658 and IM2-GC). Abbreviations: EEV, extracellular vesicles derived from erythrocytes