Fig. 1: The expression of endothelial and smooth muscle-like cell biomarkers in mouse microvascular ECs is differently regulated after irradiation.

ECs were irradiated with 15 Gy of 137Cs γ-rays and were harvested at 24, 48, and 72 h post-irradiation. A, B show representative RT-qPCR and western blot analysis of Robo4 expression levels. Data are mean ± SD; n = 3; vs. non-irradiated control. C RT-qPCR was used to measure the expression of mesenchymal cell markers after IR treatment. A marked upregulation of αSma, S100A4, SNAI1, Vimentin, and Fibronectin expression was noted following γ-radiation exposure. Data are shown as means ± SEM; n = 5. Western blot analysis demonstrated that IR increased Fibronectin, Vimentin, and αSma expression (D) and decreased PECAM-1 and VE-Cadherin (E). Data are shown as means ± SEM; n = 4. After the Robo4 gene knockdown, (F) RT-qPCR was used to evaluate the expression of mesenchymal cell markers. Bars indicate the mean fold changes ±SEM relative to the corresponding control; n = 6. G Western blots and densitometric quantification. H Immunofluorescence confocal microscopy of the extracellular matrix components (Fibronectin (FN), Vimentin, and Collagen Type1 (Col1) (red)) protein levels, respectively, in Robo4-depleted ECs. Data are mean ± SEM; n ≥ 3. I shows immunofluorescence staining and J Western blot analysis of αSma and S100A4 (green) in ECs with or without Robo4 knockdown at 48 h post-irradiation. Nuclei stained with 4’,6-diamidino-2-phenylindole (DAPI) appear blue. Values are expressed as mean ± SEM of not less than three independent experiments. Scale bars, 20 µm; *ρ < 0.05; **ρ < 0.01; ***ρ < 0.001; ****ρ < 0.0001).