Figure 5. Basal EphrinB2/EphB4 signalling controls the stability of LEC junctions through regulation of Rho-mediated cytoskeletal contractility.

Immunofluorescence of HDLECs for VE-cadherin (red) and phalloidin (green), showing increase in actin stress fibres after 3 h-treatment with the EphrinB2 blocking antibody (B11) compared to untreated controls (CTRL). Phalloidin single channel images are depicted in grey. (B) Quantification of relative central actin (total actin – junctional actin) from n = 7–11 images from three independent experiments shown in A. (C, D) Assessment of Rac1 activity in HDLECs after EphrinB2-Fc stimulation (30 min) (C) or siRNA-mediated EFNB2 silencing (48 hr) (D) Rac1-GTP-pull down and total cell lysates were immunoblotted using Rac1 antibodies. Representative Western blots are shown. Quantification was done from three independent experiments. (E) Immunofluorescence of HDLECs incubated with EphrinB2 blocking antibody (B11) for 3 hr with and without pre-incubation of the ROCK inhibitor Y-27632 using antibodies against VE-cadherin (green) and CLDN5 (grey, upper panel) or phalloidin (grey, bottom panel). Note inhibition of EphrinB2 blockade (B11)-induced junctional and cytoskeletal effects by Y-27632 treatment. (F) Quantification of relative central actin (total actin – junctional actin) from n = 6–9 images from two independent experiments shown in E. (G) Quantification of HDLEC monolayer permeability to 40 kDa FITC-dextran. Note inhibition of EphrinB2 blockade (B11)-induced increase in permeability by Y-27632 treatment. (n = 6–9 replicates per condition from three independent experiments). Data in B, F, G represent mean ± s.e.m. p value, Two-tailed unpaired Student’s t-test. Data in C, D represent mean ± s.e.m. p value, One-sample t-test. Source data for panels (C-D) are provided. Scale bars: 100 μm (A), 10 μm (E).