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Figures
- Fig. 1. Syndecan-4 mutants are located at the membrane of endothelial cells.
(A) Schemes of the syndecan-4 cytoplasmic constructs used in the study: S179A mutation expected to lead to a constitutive PKCα activation; PIP2− (Y188KK to L188QQ) mutation; PDZ− (deletion of the COOH-terminal A198 residue) mutation. (B) HUV-EC-C transfection efficiency was determined by flow cytometry on non-permeabilized cells. Transfected cells were quantified for EGFP fluorescence (horizontal axis) and for fluorescent SDC-4 staining after cell incubation with specific antibody or control isotype (vertical axis). Transfection rate from representative experiments were estimated for each plasmid. (C) Cells were transfected with GFP plasmid (control), SDC4WT-GFP (SDC4WT) or SDC-4 constructs (S179A, L188QQ or A198del) (horizontal axis). Expression of membrane SDC-4 in GFP-positive cells was quantified by flow cytometry with specific anti-SDC-4 antibodies without cell permeabilization (vertical axis). * P<0.05, versus control cells. (D) Membrane SDC-4 expression was analyzed by western blot using SDC-4 antibodies after membrane fractionation for cells transfected with SDC4WT-GFP (SDC4WT) or SDC-4 constructs (S179A, L188QQ or A198del). Specificity of SDC-4 antibodies was checked using SDC4-siRNA transfected cells (siRNA SDC-4) and siRNA negative control (SNC). (E) HUV-EC-C transfected with GFP plasmid (control) or SDC4WT-GFP (SDC4WT) were incubated with anti-β1 integrin antibodies or isotype control (red fluorescence) and analyzed under confocal microscopy (×400). Scale bars: 10 µm. The EGFP fluorescence indicates that SDC-4 is localized at the cell membrane. The immunostaining of β1 integrin was used a specific membrane cell marker. (F) Cells were transfected with GFP plasmid (control) or with SDC4WT-GFP (SDC4WT). Membrane heparan sulfate (HS) chain expression of GFP-positive cells was quantified by flow cytometry with specific anti-HS antibodies without cell permeabilization (red histogram) or with isotype control (black histogram).
- Fig. 2. Intracellular domains of syndecan-4 are involved in RANTES/CCL5-mediated HUV-EC-C migration.
(A) HUV-EC-C transfected with GFP plasmid (control) or with SDC4WT-GFP (SDC4WT) or with SDC-4 constructs (S179A, L188QQ, or A198del) were stimulated or not by 3 nM RANTES/CCL5 and cell migration was assayed by a transwell chamber model. Results are indicated as cell number/field (mean ± SEM). The vertical axis ranges from 300 to 600 cells/field. * P<0.05, RANTES/CCL5 versus control; # P<0.05, S179A versus SDC4WT (in the absence of RANTES/CCL5); $ SDC4WT versus control (in the presence of RANTES/CCL5). (B) The morphology of transfected HUV-EC-C was analyzed by live confocal microscopy upon RANTES/CCL5 stimulation for 15 minutes. (×400). Scale bars: 10 µm. Membrane protrusions were shown by white arrows.
- Fig. 3. Intracellular domains of syndecan-4 are involved in RANTES/CCL5-mediated HUV-EC-C spreading and vascular tube formation.
HUV-EC-C transfected with GFP plasmid (control), SDC4WT-GFP (SDC4WT) or mutated SDC-4 constructs (S179A, L188QQ, A198del) were stimulated or not by 3 nM RANTES/CCL5 and assayed for cell spreading on fibronectin (A) or vascular tube formation in Matrigel (B). Results are expressed as area (A) or as length of vascular sprout (B) (mean ± SEM) expressed in arbitrary units (A.U.). The vertical axis ranges from (A) 0.1 to 0.15 or from (B) 0.5 to 0.85 A.U. * P<0.05, RANTES/CCL5 versus control; # P<0.05, S179A versus SDC4 (in the absence of RANTES/CCL5); $ P<0.05 SDC4WT versus control (in the presence of RANTES/CCL5).
- Fig. 4. PKCα mediates RANTES/CCL5-induced endothelial cell migration and vascular tube formation via syndecan-4.
(A) Specificity of SDC-4 and pSDC-4 antibodies was checked using siRNA-negative control (SNC) or siRNA-SDC-4 (siRNA SDC-4) transfected cells by western blot analysis. HUV-EC-C transfected with GFP plasmid (control) or with SDC4WT-GFP (SDC4WT) were stimulated or not (U) by 3 nM RANTES/CCL5 (R) or 20 ng/ml FGF-2 (F) and SDC-4 phosphorylation at Ser179 was evaluated by western blot. Upper panel, representative Western blot analysis. Lower panel, densitometry quantification of three independent experiments. pSDC-4 band intensity was normalized to SDC-4 one. Results of relative densitometry intensities (mean ± SEM) are expressed in arbitrary units (A.U.). * P<0.05, RANTES/CCL5 or FGF-2 versus unstimulated cells. (B,C) HUV-EC-C transfected with GFP plasmid (control) or with SDC4WT-GFP (SDC4WT) were either co-transfected with a dominant negative PKCα plasmid (DN-PKCα). They were pre-incubated or not with Gö6976, a PKCα inhibitor, and treated or not with 3 nM RANTES/CCL5. They were then assayed for cell migration in a transwell chamber model (B) or for vascular tube formation in Matrigel (C). (B) Results are expressed as mean ± SEM of migrated cell number/field. Vertical axis ranges from 300 to 600 cells/field. (C) Results of vascular sprout length are presented as mean ± SEM expressed in arbitrary units (A.U.). Vertical axis ranges from 0.5 to 1 A.U. * P<0.05, RANTES/CCL5 versus untreated cells; $ P<0.05 cells treated with Gö6976 versus cells in the absence of PKCα inhibitor (in the absence of RANTES/CCL5); # P<0.05 cells preincubated with Gö6976 or with the dominant negative PKCα plasmid versus cells in the absence of PKCα inhibitor (in the presence of RANTES/CCL5).
- Fig. 5. RANTES/CCL5 induced co-localization of SDC-4 and PKCα at the cell membrane and Rac1 activation.
(A–C) HUV-EC-C were co-transfected with PKCα-DsRed2 plasmid and with either GFP plasmid (control, panels A and B) or GFP-SDC4WT (SDC4WT, panel C). They were incubated or not with (A) 0.5 µM TPA or with (B,C) 3 nM RANTES/CCL5 for 15 minutes and analyzed under live confocal microscopy. Membrane localization of SDC-4 (green) and PKCα (red) was indicated with white arrows. (×400). (D) HUV-EC-C transfected with GFP plasmid (control) or with SDC4WT-GFP (SDC4WT) or with mutated SDC-4 constructs (S179A, L188QQ, A198del) were stimulated or not (U) by 3 nM RANTES/CCL5 (R). After cell fractionation, the amount of PKCα in membrane of total fraction was evaluated by western blot. (E) HUV-EC-C transfected with GFP plasmid (control) or with SDC4WT-GFP (SDC4WT) or with mutated SDC-4 constructs (S179A, L188QQ, A198del) were stimulated or not (U) by 3 nM RANTES/CCL5 (R). Rac1-GTP activity was determined by pull down assay and analyzed using specific Rac1-GTP antibodies by western blot. Scale bars: 10 µm.
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