Supplementary Figure 4: Integrin recruitment in endothelial podosome rosettes.

(a) Confocal images of representative VEGF-stimulated EC, PMA-treated for 30 min. Inset, of the podosome-rosette. Scale bar, 20 μm. (b) Gelatin degradation assay on EC treated with rat IgG or anti-α6 blocking Ab after 1 hour of stimulation with PMA. Mean ± SEM of n = 10 cells from 3 independent experiments (^∗∗∗, P < 0.001 versus Rat IgG). Statistical significance was calculated using unpaired nonparametric Mann-Whitney test. (c) Graph shows the percentages of individual podosome positive EC, stimulated as indicated and treated with rat IgG or anti-α6 blocking Ab. Mean ± SEM of n = 3 independent experiments in which 250 total cells were analyzed cells per experimental point. (d) Cytofluorimetric analysis of membrane integrin α6 localization. EC were transduced with shRNA scramble (shSCRL) or against integrin α6 (shITGA6_4 and shITGA6_5). Mean ± SEM of n = 3 independent experiments (^∗∗∗, P < 0.001 versus shSCRL). Statistical significance was calculated using one-way ANOVA test followed by Bonferroni adjusted post-hoc t-tests. (e) Cytofluorimetric analysis of membrane integrin α6 localization. EC were incubated for 24 h in M199 10% FCS (unstimulated) or in M199 10% FCS plus 30 ng/ml of VEGF-A (24h VEGF-A) or for 48h in M199 10% FCS plus 30 ng/ml of VEGF-A (48h VEGF-A). Mean ± SEM of n = 3 independent experiments. (^∗∗, P < 0.01 versus unstimulated; ^∗∗∗, P < 0.001). Statistical significance was calculated using one-way ANOVA test followed by Bonferroni adjusted post-hoc t-tests.