Extended Data Fig. 2
From: Syndecan-2 selectively regulates VEGF-induced vascular permeability
a, confocal image of confluent HUVEC cells in basal conditions showing human DEP1 carrying N-terminal HA-tag (DEP1-HA, red) expression at cell–cell junctions labeled with VE-cadherin (cyan), white arrows, lower panels. Scale bar: 25 μm. b, confocal image of HUVEC cells in basal conditions expressing both human DEP1 carrying N-terminal HA-tag (DEP1-HA, red) and human Sdc2 carrying N-terminal SNAP-tag (SDC2-SNAP, green) proteins. Lower panels show magnification of box in upper panels showing localization of both proteins at the plasma membrane and at the membrane protrusions. Scale bar: 25 μm. c, f, SIM imaging of SDC2/DEP1/Rab5 and SDC2/DEP1/VEGFR2 complexes in HUVECs expressing DEP1-HA and SDC2-SNAP proteins and treated for 20 min with 50 ng/ml VEGFA165. Scale bar: 0.5 μm. d,f, line profile analysis illustrating the distance between DEP1, Sdc2 and Rab5 or DEP1, Sdc2 and VEGFR2 in the same compartment. Plots represent the fluorescent signal in SIM images as a function of position along the line profile in left panel (red dotted line). g, h confocal images of constitutive internalization of DEP1 after incubation for 0 min, 30 min and 60 min in absence of VEGFA in HUVECs control and HUVECs transfected with Sdc2 siRNA. Scale bar: 15 μm. i, confocal image of internalized SDC2–DEP1 complexes in Rab5+ endosomes (white arrows) following constitutive endocytosis in VEGFA-free media. Scale bar: 5 μm. All images are representative images of >3 independent experiments (n). Data are presented as mean values + /- SEM (standard error of the mean). Statistical analysis was performed by two-way ANOVA with Sidak’s multiple comparison test (panels h), (N.S. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001).
