Figure 5: Arf6 is required for HGF-induced β1 integrin recycling/activation in endothelial cells.

(a,b) Effects of Arf6 deletion on surface β1 integrin level in HGF- and VEGF-stimulated iECs. After stimulation of cells with HGF (a) and VEGF (b) for the indicated times, β1 integrin levels at the plasma membrane of control and Arf6^−/− iECs were detected (upper panels) as described in Methods section. The data were quantified and represented as means±s.e.m. from three independent experiments (lower panels). (c) Effects of Arf6 deletion on HGF-induced recycling of β1 integrin from internal compartments to the plasma membrane in iECs. Internal β1 integrin labelled with anti-β1 integrin antibody (HMβ1-1) were detected by immunostaining after stimulation of cells with or without HGF and following wash out of cell surface antibody (c, left), and its fluorescence intensity was measured (c, right). Means±s.e.m. from four independent experiments were shown. Scale bar, 20 μm. (d) Effects of Arf6 deletion on HGF-induced internalization of β1 integrin by iECs. Biotin-labelled surface β1 integrin were internalized by incubating with or without HGF and internal β1 integrin were detected as described in Methods section. Means±s.e.m. from three independent experiments were shown. (e) Inhibition of HGF-induced β1 integrin recycling/activation by Arf6 deletion from iECs. The active form of β1 integrin was immunostained with the antibody 9EG7 (green; e, left), and its intensity from three independent experiments was measured for >50 cells (e, right; median, quartiles, maximum and minimum values are indicated). Cells were also stained with DAPI (blue). Scale bar, 20 μm. (f) Immunostaining for active form of β1 integrin in blood vessels of B16 melanoma tumours produced in control and EC-Arf6 cKO mice. Scale bar, 50 μm. *P<0.05; NS, not significant, Student’s t-test for c and non-parametric Mann–Whitney U-test for e.