Supplementary Figure 5: Integrin α6 recruitment in endothelial podosomes and integrin α6 silencing in aortic explants.

(a) Integrin α6 fluorescence quantification in rosettes regions. Rosettes ROI were manually selected using colocalization of cortactin and F-actin staining. Mean ± SEM of n = 3 independent experiments in which 30 total cells were analyzed cells per experimental point. (^∗∗∗, P < 0.001 versus T = 0). Statistical significance was calculated using paired nonparametric Wilconox test. (b) TIRF microscopy of LifeAct-RFP (red) and α6-GFP (green) localization in EC treated with PMA for 15 min. EC were seeded on gelatin-coated glass-bottom dishes. The complete sequence of time-lapse TIRF microscopy is shown in Video S2. Scale bar, 20 μm. Zoom of white dotted square is shown in the lower panels. Scale bar, 1 μm. (c) Endothelial layer of a 48 hours VEGF-A-stimulated aortic explant from Fig. 3F. In yellow, 3D reconstruction of the co-localization channel in podosome rosette. Scale bar: 20 m. (d) Aortic explants were incubated for 48 hours in M199 10% FCS (unstimulated) or M199 10% FCS with 30 ng/ml of VEGF-A (48 h VEGF-A) plus lentiviruses carrying scramble shRNA (SCRL shRNA) and shRNA targeting murine ITGA6 (shRNA48 and shRNA50). Graph shows the percentage (%) of podosome-rosettes positive in the endothelial layer of aortic explants, treated and transduced as indicated. Mean ± SEM of n = 3 independent experiments in which 340 total nuclei were analyzed cells per experimental point. (^°°, P < 0.01 versus unstimulated; ^∗, P < 0.05 versus SCRL shRNA; ^∗∗, P < 0.01 versus SCRL shRNA.) Statistical significance was calculated using one-way ANOVA test followed by Bonferroni adjusted post-hoc t-tests.