Figure 4.

EC-MVs promote stress fibre formation, MLC, and VE-cadherin phosphorylation. (A–C) Representative confocal microscopy images and z stack analysis showing increased F-actin stress fibres (arrowheads) in ECs after EC-MV interaction. (D–F) The actin-binding protein cortactin is increased and redistributes from the cell periphery to more centralized intracellular locations after EC-MV treatment. Scale bar corresponds to 10µm and applies to all images.**P < 0.01 between buffer and EC-MV treated cells by Student’s t-test, n = 10–12 different confocal z stacks from multiple slides. (G and H) Representative confocal microscopy images showing EC-MV interaction with ECs increase pMLC2 (Thr18/Ser19) which co-stains with F actin (arrowheads). (I) A three-dimensional reconstruction of Panel H reveal MVs fused to the cell membrane as well as inside the cell (arrowheads) at the same focal plane with F-actin stress fibres and pMLC2. (J and K) Representative confocal microscopy images showing EC-MV interaction increases Tyr⁶⁵⁸ p-VE-Cadherin compared to control cells. (L and M) Representative western blot images and analysis showing increased percentage of phosphorylated MLC2 and VE-cadherin after EC-MV treatment. All data are shown as mean ± SEM. **P < 0.01 by Student’s t-test, n = 8 lysates.