Ensity followed by normalization with regard to tubulin and expressed as a fold change compared with the control (no galectin addition).Proximity ligation assayWe used the Duolink in situ PLA kit from Olink Bioscience (Olink Bioscience, Uppsala, Sweden) to detect colocalisation between VEGFR1 or VEGFR2 and early endosome antigen-1 (EEA1) according to the manufacturer’s instructions (Materials and Methods S1). The PLA signal/cell was determined with image analysis software developed by the Laboratory of Image Synthesis and Analysis (ULB, Brussels, Belgium) (Materials and Methods S1). Each condition was evaluated in two independent experiments.Figure 4. Galectin-induced activation of ERK1/2 and Hsp27. Determination of ERK1/2 (A, C) and Hsp27 (B, D) phosphorylation levels following a 10-min stimulation of EA.hy926 cells with galectin-1, galectin-3 or both galectins (1 mg/ml each), by ELISA (A, B) and Western blots (C, D). For ELISAs, 11967625 the data (mean +/2 SEM) are shown as relative values compared with the control (no galectin addition), and significant differences are indicated (* p,0.05, ** p,0.01 and *** p,0.001). Quantification of Western blots was done using ImageJ (see Materials and Methods). doi:10.1371/journal.pone.0067029.gVEGFR Involvement in Galectin-Induced AngiogenesisFigure 5. Modulation of VEGFR endocytosis by exogenous galectins in EA. hy926 cells. The effects of exogenous galectins (1 mg/ml each) were evaluated by analysing the colocalisation between each receptor 1315463 and EEA1 using the proximity ligation assay and an image analysis tool. Representative images of z-stacks of 7 fluorescent micrographs projected into a single phase-contrast image (original magnification: 660) are shown. Signal/cell values are shown as relative values (mean +/2 SEM) compared with the control (no galectin addition). The tables show the significance levels obtained by applying the standard Dunn procedure (post-hoc test) to compare all the pairs of experimental conditions, in order to avoid multiple comparison effects (NS = not significant: p.0.05). Scale bar: 20 mm. doi:10.1371/journal.pone.0067029.gStatistical analysesThe non-parametric Kruskal-Wallis test was used to compare multiple independent Title Loaded From File groups of numerical data. If the test was significant, post-hoc tests were applied using either the standard Dunn procedure to compare all group pairs or its adaptation to compare each experimental condition to the control, avoiding multiple comparison effects (as detailed in Zar [25]). To evaluate whether the combined effect induced by the two galectins was additive or synergistic (the latter being defined as a total effect greater than the sum of the Sion with oxygen-supplemented medium, the collected urine was loaded onto 20612-cm individual effects), we used the adjusted rank transform test described by Leys et al [26]. All statistical analyses were performed using Statistica (Statsoft, Tulsa, OK, USA).(10 mg/ml). The addition of both galectins together (10 mg/ml each) to the culture medium increased cell growth to a similar level as galectin-1 alone (Figure 1B).Modulation of tube formation by exogenous galectinsIn both EA.hy926 cells and HUVECs, the addition of galectin-1 or galectin-3 alone stimulated tube formation (Figure 2). Regarding EA.hy926 cells, the addition of both galectins together at 1 mg/ml each induced a significant and synergistic effect on the total tube length (average tube length increase of 25 , 23 and 94 in response to galectin-1, galectin-3 and galectin-1+ galectin3, respectively) (Figures 2A, C), paralleled by.Ensity followed by normalization with regard to tubulin and expressed as a fold change compared with the control (no galectin addition).Proximity ligation assayWe used the Duolink in situ PLA kit from Olink Bioscience (Olink Bioscience, Uppsala, Sweden) to detect colocalisation between VEGFR1 or VEGFR2 and early endosome antigen-1 (EEA1) according to the manufacturer’s instructions (Materials and Methods S1). The PLA signal/cell was determined with image analysis software developed by the Laboratory of Image Synthesis and Analysis (ULB, Brussels, Belgium) (Materials and Methods S1). Each condition was evaluated in two independent experiments.Figure 4. Galectin-induced activation of ERK1/2 and Hsp27. Determination of ERK1/2 (A, C) and Hsp27 (B, D) phosphorylation levels following a 10-min stimulation of EA.hy926 cells with galectin-1, galectin-3 or both galectins (1 mg/ml each), by ELISA (A, B) and Western blots (C, D). For ELISAs, 11967625 the data (mean +/2 SEM) are shown as relative values compared with the control (no galectin addition), and significant differences are indicated (* p,0.05, ** p,0.01 and *** p,0.001). Quantification of Western blots was done using ImageJ (see Materials and Methods). doi:10.1371/journal.pone.0067029.gVEGFR Involvement in Galectin-Induced AngiogenesisFigure 5. Modulation of VEGFR endocytosis by exogenous galectins in EA. hy926 cells. The effects of exogenous galectins (1 mg/ml each) were evaluated by analysing the colocalisation between each receptor 1315463 and EEA1 using the proximity ligation assay and an image analysis tool. Representative images of z-stacks of 7 fluorescent micrographs projected into a single phase-contrast image (original magnification: 660) are shown. Signal/cell values are shown as relative values (mean +/2 SEM) compared with the control (no galectin addition). The tables show the significance levels obtained by applying the standard Dunn procedure (post-hoc test) to compare all the pairs of experimental conditions, in order to avoid multiple comparison effects (NS = not significant: p.0.05). Scale bar: 20 mm. doi:10.1371/journal.pone.0067029.gStatistical analysesThe non-parametric Kruskal-Wallis test was used to compare multiple independent groups of numerical data. If the test was significant, post-hoc tests were applied using either the standard Dunn procedure to compare all group pairs or its adaptation to compare each experimental condition to the control, avoiding multiple comparison effects (as detailed in Zar [25]). To evaluate whether the combined effect induced by the two galectins was additive or synergistic (the latter being defined as a total effect greater than the sum of the individual effects), we used the adjusted rank transform test described by Leys et al [26]. All statistical analyses were performed using Statistica (Statsoft, Tulsa, OK, USA).(10 mg/ml). The addition of both galectins together (10 mg/ml each) to the culture medium increased cell growth to a similar level as galectin-1 alone (Figure 1B).Modulation of tube formation by exogenous galectinsIn both EA.hy926 cells and HUVECs, the addition of galectin-1 or galectin-3 alone stimulated tube formation (Figure 2). Regarding EA.hy926 cells, the addition of both galectins together at 1 mg/ml each induced a significant and synergistic effect on the total tube length (average tube length increase of 25 , 23 and 94 in response to galectin-1, galectin-3 and galectin-1+ galectin3, respectively) (Figures 2A, C), paralleled by.
