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Fig. 4 | Cellular & Molecular Biology Letters

Fig. 4

From: Robo4 inhibits gamma radiation-induced permeability of a murine microvascular endothelial cell by regulating the junctions

Fig. 4

Overexpression of Robo4 improves IR-induced downregulation of VE-cadherin and decreases the permeability of the EC monolayer. A Confirmation of Robo4 overexpression after establishing a stable cell line using western blot and qPCR analysis, respectively. B Effect of Robo4 overexpression and irradiation on cell survival and proliferation of ECs. C and E Functional analysis of Robo4 overexpression by FITC-dextran (70 kDa) permeability assay and EC monolayer crystal violet staining after irradiation. D Western blot analysis of total and pY-731 VE-cadherin after Robo4 overexpression and irradiation; the relative protein expression of phosphorylated Y-731 and total VE-cadherin normalized with endogenous beta-actin protein; The ratio of pY-731 VE-cadherin to total VE-cadherin. 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 DAPI (blue). H Western blot analysis showing connexin 43 expression levels in Robo4 overexpression microvascular ECs after gamma radiation treatment. J Confocal images show immunofluorescence of connexin 43 (red) in Robo4 overexpression microvascular ECs, with or without irradiation. Nuclei appear in blue. I Flow cytometric analysis to determine EC GJ coupling ability in vitro. For all experiments, scale bars = 20 µm, n ≥ 3, and error bars represent std. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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