A significant effect on branching point numbers (average branching point number increase of 36 , 67 and 195 in response to galectin-1, galectin-3 and galectin-1+ galectin-3, respectively) (Figure 2E). This 1317923 effect was not statistically detected as synergistic, probably because of a larger heterogeneity in these data compared with the tube length. The addition of both galectins at 10 mg/ml each had an antagonistic effect on tube length and branching points (average tube length increase of 47 and 50 and average branching point number increase of 106 and 79 in response to galectin-1 and galectin-3, respectively, in contrast to an average tube length Thiazole Orange site decrease of 31 and an average branching point number increase of only 7 in response to galectin-1+ galectin-3) (Figures 2C, E).Results Modulation of cell growth by exogenous galectinsWe observed different responses to exogenous galectins depending on the cell line. In EA.hy926 cells, we observed no significant effect of galectin-1 or galectin-3 (Figure 1A). In contrast, the addition of both galectins together at 10 mg/ml each increased cell growth by 43 (p,0.01). Galectin-1 alone significantly increased HUVEC growth at 48 h by 47 (p,0.001; Figure 1B). A slight but not statistically significant increase (12 ) was also observed for galectin-3 aloneVEGFR Involvement in Galectin-Induced AngiogenesisIn contrast, in HUVECs, an additive effect on tube length and branching point number was induced by the addition of both galectins 1315463 at 10 mg/ml each (average tube length increase of 41 , 36 and 87 and average branching point number increase of 41 , 38 and 87 in response to galectin-1, galectin-3 and galectin-1+ galectin-3, respectively) (Figures 2B, D, F).galectin-3 and galectin-1+ galectin-3, respectively estern blot quantification was not possible due to absence of phosphorylation in the control condition) and HSp27 (average increase of 25 , 39 and 55 evaluated by ELISA and fold change of 6.7, 9.3 and 15.1 evaluated by Western blots in response to galectin-1, galectin3 and galectin-1+ galectin-3, respectively).Galectin-induced tube formation is related to VEGFR activationBased on the EC response to galectins, we next investigated the enhanced effect induced by galectin-1 and galectin-3 added together at 1 mg/ml each on EA.hy926 cells. We determined the pathways underlying this galectin-induced stimulation. Previous studies have shown that galectin-1 and galectin-3 can activate VEGFR2 [3,4]. However, we did not find any data in the literature (to the best of our knowledge) related to the galectininduced activation of VEGFR1. Title Loaded From File Therefore, we analysed the expression and phosphorylation levels (by ELISA and western blotting) of VEGFR2 and VEGFR1 in EA.hy926 cells after galectin stimulation. The addition of galectin-1, galectin-3 or both galectins together had no effect on VEGFR1 or VEGFR2 protein expression (Figures 3C ). As shown in Figs. 3A , galectin-1 and galectin-3 alone induced VEGFR2 phosphorylation without VEGFR1 phosphorylation. In contrast, the addition of both galectins together induced VEGFR1 and VEGFR2 phosphorylation. VEGFR2 activation was inhibited by lactose but not sucrose, indicating that the effect is due to glycan binding by galectins (Figure S2). Next, we examined whether the galectin-induced activation of VEGFRs was involved in galectin-induced tube formation. For this purpose, we added either blocking VEGFR1 Ab or blocking VEGFR2 Ab to EA.hy926 cells plated on matrigel.A significant effect on branching point numbers (average branching point number increase of 36 , 67 and 195 in response to galectin-1, galectin-3 and galectin-1+ galectin-3, respectively) (Figure 2E). This 1317923 effect was not statistically detected as synergistic, probably because of a larger heterogeneity in these data compared with the tube length. The addition of both galectins at 10 mg/ml each had an antagonistic effect on tube length and branching points (average tube length increase of 47 and 50 and average branching point number increase of 106 and 79 in response to galectin-1 and galectin-3, respectively, in contrast to an average tube length decrease of 31 and an average branching point number increase of only 7 in response to galectin-1+ galectin-3) (Figures 2C, E).Results Modulation of cell growth by exogenous galectinsWe observed different responses to exogenous galectins depending on the cell line. In EA.hy926 cells, we observed no significant effect of galectin-1 or galectin-3 (Figure 1A). In contrast, the addition of both galectins together at 10 mg/ml each increased cell growth by 43 (p,0.01). Galectin-1 alone significantly increased HUVEC growth at 48 h by 47 (p,0.001; Figure 1B). A slight but not statistically significant increase (12 ) was also observed for galectin-3 aloneVEGFR Involvement in Galectin-Induced AngiogenesisIn contrast, in HUVECs, an additive effect on tube length and branching point number was induced by the addition of both galectins 1315463 at 10 mg/ml each (average tube length increase of 41 , 36 and 87 and average branching point number increase of 41 , 38 and 87 in response to galectin-1, galectin-3 and galectin-1+ galectin-3, respectively) (Figures 2B, D, F).galectin-3 and galectin-1+ galectin-3, respectively estern blot quantification was not possible due to absence of phosphorylation in the control condition) and HSp27 (average increase of 25 , 39 and 55 evaluated by ELISA and fold change of 6.7, 9.3 and 15.1 evaluated by Western blots in response to galectin-1, galectin3 and galectin-1+ galectin-3, respectively).Galectin-induced tube formation is related to VEGFR activationBased on the EC response to galectins, we next investigated the enhanced effect induced by galectin-1 and galectin-3 added together at 1 mg/ml each on EA.hy926 cells. We determined the pathways underlying this galectin-induced stimulation. Previous studies have shown that galectin-1 and galectin-3 can activate VEGFR2 [3,4]. However, we did not find any data in the literature (to the best of our knowledge) related to the galectininduced activation of VEGFR1. Therefore, we analysed the expression and phosphorylation levels (by ELISA and western blotting) of VEGFR2 and VEGFR1 in EA.hy926 cells after galectin stimulation. The addition of galectin-1, galectin-3 or both galectins together had no effect on VEGFR1 or VEGFR2 protein expression (Figures 3C ). As shown in Figs. 3A , galectin-1 and galectin-3 alone induced VEGFR2 phosphorylation without VEGFR1 phosphorylation. In contrast, the addition of both galectins together induced VEGFR1 and VEGFR2 phosphorylation. VEGFR2 activation was inhibited by lactose but not sucrose, indicating that the effect is due to glycan binding by galectins (Figure S2). Next, we examined whether the galectin-induced activation of VEGFRs was involved in galectin-induced tube formation. For this purpose, we added either blocking VEGFR1 Ab or blocking VEGFR2 Ab to EA.hy926 cells plated on matrigel.
