Fig. 5

Irradiation-induced dislocalization and degradation of VE-cadherin and VEGF-A-induced activation of ADAM10. a–d) Immunofluorescence stainings showing subcellular distribution of VE-cadherin in endothelial cells grown on coverslips. Upon reaching confluence, cells were mock-irradiated (a), irradiated with 4 Gy (b and C), or treated with 100 ng/ml VEGF-A (d) and prepared for VE-cadherin (green; Hoechst-33,342 nuclear staining is shown in blue) immunofluorescence microscopy after 2 h (B and D) or 24 h (C; 4 Gy only). Arrowheads indicate weakened or absent VE-cadherin staining at cell-cell contact sites. Asterisks mark areas of granular VE-cadherin staining indicating dislocation from cell-cell contact sites. E–H) VE-cadherin localization in control and 4 Gy-irradiated endothelial cell layers in the absence or presence of the ADAM10-inhibitor GI254023X (10 μM). Cells were fixed and stained for VE-cadherin (green; nuclei are blue) after 24 h. Scale bars in A–H, 20 μm. I) ADAM10 expression (precursor and mature form) in endothelial cells treated with irradiation (4 Gy; proteins isolated after 24 h) or VEGF-A (100 ng/ml; proteins isolated after 4 h) in the absence or presence of GI253023X (10 μM; added 30 min before treatments). Data (n ≥ 3) are shown as means ± standard deviations. Statistics: t-test, *p < 0.05, **p < 0.01, ***p < 0.001