Figure 5.

EC-MVs deficient in c-Src have reduced effects on albumin flux, stress fibre formation, MLC, and VE-cadherin phosphorylation. (A) EC-MVs derived from c-Src knockdown HUVECs display negligible c-Src compared to those from scrambled siRNA-treated cells, confirming the efficiency of c-Src knockdown. (B) Western blot image showing knockdown of intracellular c-Src in ECs after transfection with c-Src siRNA. (C) Albumin permeability through endothelial monolayers is increased at 16 h after treatment with scrambled EC-MVs but not by c-Src deficient EC-MVs. *P < 0.05, **P < 0.01 by one-way ANOVA with Tukey’s multiple comparison tests, n = 6 experiments. (D–F) Confocal microscopy z stack analysis comparing F-actin levels in endothelial monolayers treated with scrambled vs. Src-deficient EC-MVs. (G–I) Comparison of cortactin levels from confocal z stacks in endothelial cells treated with scrambled vs. Src-deficient EC-MVs. *P < 0.05, **P < 0.01 by Student’s t-test, n = 10–18 different confocal z stacks from multiple slides. Representative confocal images showing increased phospho-MLC levels (J and K) and Tyr⁶⁵⁸ p-VE-Cadherin levels (M and N) in endothelial cells treated with scrambled vs. Src-deficient EC-MVs. Scale bar corresponds to 10µm and applies to all images. (L, O) Western blot images and analysis showing increased percentage of pMLC2 and Tyr⁶⁵⁸ p-VE-Cadherin in endothelial cells treated with scrambled vs. Src-deficient EC-MVs. Data are shown as mean ± SEM. **P < 0.01 by Student’s t-test, n = 5–8 lysates.