Fig. 4

Oral cancer cell-derived EVs induce vascular destabilization. a Analysis of the gap area in HUAEC monolayer staining images. The gap area was determined by detecting the black (unstained) area in the cell layers and defined by the boundaries between black (unstained area) and VE-cadherin-positive area. b HUAEC monolayers cultured on cover slip were treated with vehicle (PBS) or EVs (Ctrl-EVs or Tβ-EVs) for 72 h. Confocal images showing the localization of VE-cadherin in HUAECs. Cells were fixed and stained with anti-VE-cadherin (green) and Hoechst33342 (nuclei: blue). The red dot line indicates the gap edge, and the yellow filled-in area indicates the gap area. Scale bars, 50 μm. c Quantification of the gap area. The gaps were quantified in five fields of view from at least four independent samples and represented as the percentage of formed gaps in the total area. d HUAEC monolayers cultured on transwell with 1-μm pore were treated with vehicle (PBS) or EVs (Ctrl-EVs or Tβ-EVs) for 72 h. Analysis of the vascular destabilization as the increase in HUAEC permeability. Permeability was analyzed by measurement of FITC-dextran (70 kDa) leakage into the lower well. Data are represented as the mean ± SD from three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 by ordinary one-way ANOVA with Tukey’s multiple comparisons test