Fig. 3: Inhibition of IR-induced Endo-MT upon Robo4 overexpression in ECs.

A Western blotting for the components of the ECM expression in Robo4 overexpressed murine microvascular ECs, 2 days after radiation exposure to 15 Gy. The bars represent mean ± SEM; n = 3. B Immunofluorescence staining analysis by Confocal microscopy of ECM components (Fibronectin (FN) and Collagen Type1 (Col1) (red)) and (C) other mesenchymal markers (αSma and S100A4 (green)) protein expression in irradiated or unirradiated ECs with or without Robo4 overexpression. Nuclei appear blue (DAPI), and Scale bars represent 20 µm. D Relative expression of the smooth muscle-like cell markers: αSma and S100A4 detected by western blotting under 15 Gy dose of γ-radiation following lentiviral-mediated Robo4 upregulation. β-actin was used as an internal control. The graphs represent means ± SEM of at least three independent experiments. E Western blot and F immunocytochemistry were performed to detect the protein levels of endothelial-specific biomarkers (VE-Cadherin, PECAM-1, TIE-2, and Claudin-5) in mice microvascular ECs treated with or without 15 Gy γ-radiation upon Robo4-enhanced expression. Representative images and quantitative histograms indicated mean ± SEM; n ≥ 3. G Immunoblotting and qPCR analysis showing VCAM-1 and ICAM-1 expression levels in Robo4 overexpressed microvascular ECs following gamma radiation treatment. Data are mean ± SEM; n ≥ 3. Scale bars, 20 µm; *ρ < 0.05; **ρ < 0.01; ***ρ < 0.001; ****ρ < 0.0001).