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. 2016 Nov 30;7:13515. doi: 10.1038/ncomms13515

Figure 6. Net4 activity on angiogenesis is dependent on Net4/laminin interaction.

Figure 6

(a) Analyses of tube-like structure inhibition by Net4-ΔC mutants (Net4-ΔCE195A, Net4-ΔCR199A and Net4-ΔCΔKAPGA) impaired in laminin γ1 binding (protein concentrations: 1 μM). Statistical analyses of tube formation via determining the number of cell cluster connections (tube number) (mean±s.d.; n=3; ***P=0.0007) (right). Error bars, s.d. (n=3 independent cell cultures). (b) Blocking of Net4-ΔC inhibited tube formation through equimolar addition of γ1LN-LEa1-4 (1 μM, molar ratio of Net4-ΔC:γ1LN-LEa1-4, 1:1). The laminin fragment γ1LN-LEa1-4 competes with Net4 binding to laminin (mean±s.d.; n=3; *P=0.035; ns, not significant). Error bars, s.d. (n=3 independent cell cultures). (c) Treatment of VEGF-A induced spheroid sprouting of HDMECs embedded in collagen I by Net4-ΔC (1 μM). Analysis of sprouting events (ns, not significant). (d) Tube formation analyses of co-cultured endothelial and perivascular-like cells from untreated and Net4-FL- or Net4-FLE195A,R199A-treated cultures (30 nM). Representative images of CD31 staining are shown. The tube length (mm per mm2) was quantified through the CD31 staining (mean±s.d.; n=5; ****P<0.00005). (e) Apotome images of Col-IV, Lmγ1 and CD31 stained tubes from control (ctrl), Net4-FL and Net4-FLE195A,R199A treatment are displayed. Error bars, s.d. (n=3, (a–c); n=5, (d,e) independent cell cultures). P values, two sided t-test. Scale bars, 100 μm (a–d); 50 μm (e).