Fig. 2

Robo4 knockdown depletes the VE-cadherin expression and increases EC monolayer permeability following irradiation. Establishment of a microvascular ECs line with Robo4 stable knockdown. A Western blot and RT-qPCR analysis to confirm the reduced expression of Robo4 using beta-actin as an endogenous control. B Effect of Robo4 silencing and irradiation on cell survival and proliferation of ECs. C and E Fitc-labeled dextran-40 kDa permeability assay and Transwell endothelial monolayer crystal violet staining results following Robo4 knockdown and gamma radiation treatment. D Immunoblotting analysis of total and phosphorylated Y-731 VE-cadherin in Robo4 silenced microvascular ECs after irradiation; The relative protein expression of phosphorylated Y-731 and total VE-cadherin normalized with endogenous beta-actin protein; the ratio of phosphorylated Y-731 VE-cadherin to total VE-cadherin using image J software for quantification. Cycloheximide treatment and immunoblotting were conducted as in (F). The CD144 band intensity was normalized to actin and then normalized to the t = 0 controls. G Confocal microscopic images of VE-cadherin (red) and 4’,6-diamidino-2-phenylindole (DAPI) staining (blue). H Western blot and qPCR analysis showing connexin 43 expression levels in Robo4 knockdown microvascular ECs following gamma radiation treatment. I Confocal images show immunofluorescence of connexin 43 (red) in Robo4 knockdown microvascular ECs, with or without irradiation. Nuclei appear in blue. Scale bars = 20 µm. J Flow cytometric analysis to determine endothelial GJs coupling ability in vitro. For all experiments, n ≥ 3, and error bars represent std. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001