Addition), and significant differences are indicated (* p,0.05, ** p,0.01 and *** p,0.001). doi:10.1371/journal.pone.0067029.gVEGFR Involvement in Galectin-Induced AngiogenesisFigure 2. Modulation of tube formation by exogenous galectins. EA.hy926 (A, C, E) and HUVEC (B, D, F) cells were suspended in complete medium in 1480666 the presence or Ivation of the MAPK signaling pathway plays a pivotal role in absence of galectins at the indicated concentrations and seeded on top of matrigel layers. Representative images obtained at 22 h for EA.hy926 (A) and 6 h for HUVEC (B) are shown. Tube formation was quantified by measuring the total length of the tube network (C, D) or by counting branching point (E, F) in EA.hy926 cells (C, E) and HUVECs (D, F). 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). Scale 1527786 bar: 300 mm. doi:10.1371/journal.pone.0067029.gVEGFR Involvement in Galectin-Induced AngiogenesisFigure 3. Effects of exogenous galectins on VEGFR activation and involvement of VEGFRs in galectin-induced tube formation. (A ) Determination of VEGFR1 (A, C) and VEGFR2 (B, D) phosphorylation levels following a 5-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, 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 WesternVEGFR Involvement in Galectin-Induced Angiogenesisblots was done using ImageJ (see Materials and Methods). (E ) EA.hy926 cells were suspended in complete medium in the presence or absence of galectins (1 mg/ml each) and blocking VEGFR1 Ab (5 mg/ml) or control IgG (5 mg/ml) (E, G) or blocking VEGFR2 Ab (50 ng/ml) or control IgG (50 ng/ ml) (F, H) and seeded on top of matrigel layers. Tube formation was quantified by measuring the total length of the tube network (E ) or counting branching Title Loaded From File points (G ). The data (mean +/2 SEM) are shown as relative values compared with the control (without the addition of galectins or an inhibitor). Significant differences are indicated on horizontal arrows (the same galectin-related conditions were compared in the absence or presence of a blocking Ab using the Mann-Whitney test. * p,0.05, ** p,0.01 and *** p,0.001). doi:10.1371/journal.pone.0067029.gWestern blotsEA.hy926 lysates were analyzed by Western blots, as previously detailed [23]. Total and phosphorylated protein expression levels were evidenced by means of specific antihuman Abs against VEGFR1 (Abcam, 1/1000), phosphoVEGFR1 (R Dsytems, 1 mg/ml), VEGFR2 (Cell Signaling, Beverly, MA, 1/1000), phospho-VEGFR2 (Cell Signaling, 1/ 500), ERK 1/2(R Dsytems, 0.5 mg/ml), phospho-ERK 1/2 (R Dsytems, 0.1 mg/ml), Hsp27 (R Dsytems, 0.1 mg/ml), phospho-Hsp27(R Dsytems, 0.1 mg/ml), FAK (R Dsytems, 1 mg/ml), phospho-FAK (R Dsytems, 2 mg/ml), Src (R Dsytems, 0.1 mg/ml), phospho-Src (R Dsytem, 1 mg/ml), Akt (R Dsytems, 1 mg/ml) and phospho-Akt (R Dsytems, 1 mg/ ml). Evaluation of total proteins was performed on the membranes corresponding to their phosphorylated forms after stripping using Restore Western Blot Stripping Buffer (Thermo Scientific) according to the manufactorer’s protocol. Themonoclonal anti-tubulin Ab (Abcam,1/5000) was used as loading control. Quantification of Western blots was done using ImageJ software by integrating the band int.Addition), and significant differences are indicated (* p,0.05, ** p,0.01 and *** p,0.001). doi:10.1371/journal.pone.0067029.gVEGFR Involvement in Galectin-Induced AngiogenesisFigure 2. Modulation of tube formation by exogenous galectins. EA.hy926 (A, C, E) and HUVEC (B, D, F) cells were suspended in complete medium in 1480666 the presence or absence of galectins at the indicated concentrations and seeded on top of matrigel layers. Representative images obtained at 22 h for EA.hy926 (A) and 6 h for HUVEC (B) are shown. Tube formation was quantified by measuring the total length of the tube network (C, D) or by counting branching point (E, F) in EA.hy926 cells (C, E) and HUVECs (D, F). 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). Scale 1527786 bar: 300 mm. doi:10.1371/journal.pone.0067029.gVEGFR Involvement in Galectin-Induced AngiogenesisFigure 3. Effects of exogenous galectins on VEGFR activation and involvement of VEGFRs in galectin-induced tube formation. (A ) Determination of VEGFR1 (A, C) and VEGFR2 (B, D) phosphorylation levels following a 5-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, 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 WesternVEGFR Involvement in Galectin-Induced Angiogenesisblots was done using ImageJ (see Materials and Methods). (E ) EA.hy926 cells were suspended in complete medium in the presence or absence of galectins (1 mg/ml each) and blocking VEGFR1 Ab (5 mg/ml) or control IgG (5 mg/ml) (E, G) or blocking VEGFR2 Ab (50 ng/ml) or control IgG (50 ng/ ml) (F, H) and seeded on top of matrigel layers. Tube formation was quantified by measuring the total length of the tube network (E ) or counting branching points (G ). The data (mean +/2 SEM) are shown as relative values compared with the control (without the addition of galectins or an inhibitor). Significant differences are indicated on horizontal arrows (the same galectin-related conditions were compared in the absence or presence of a blocking Ab using the Mann-Whitney test. * p,0.05, ** p,0.01 and *** p,0.001). doi:10.1371/journal.pone.0067029.gWestern blotsEA.hy926 lysates were analyzed by Western blots, as previously detailed [23]. Total and phosphorylated protein expression levels were evidenced by means of specific antihuman Abs against VEGFR1 (Abcam, 1/1000), phosphoVEGFR1 (R Dsytems, 1 mg/ml), VEGFR2 (Cell Signaling, Beverly, MA, 1/1000), phospho-VEGFR2 (Cell Signaling, 1/ 500), ERK 1/2(R Dsytems, 0.5 mg/ml), phospho-ERK 1/2 (R Dsytems, 0.1 mg/ml), Hsp27 (R Dsytems, 0.1 mg/ml), phospho-Hsp27(R Dsytems, 0.1 mg/ml), FAK (R Dsytems, 1 mg/ml), phospho-FAK (R Dsytems, 2 mg/ml), Src (R Dsytems, 0.1 mg/ml), phospho-Src (R Dsytem, 1 mg/ml), Akt (R Dsytems, 1 mg/ml) and phospho-Akt (R Dsytems, 1 mg/ ml). Evaluation of total proteins was performed on the membranes corresponding to their phosphorylated forms after stripping using Restore Western Blot Stripping Buffer (Thermo Scientific) according to the manufactorer’s protocol. Themonoclonal anti-tubulin Ab (Abcam,1/5000) was used as loading control. Quantification of Western blots was done using ImageJ software by integrating the band int.
