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. Author manuscript; available in PMC: 2010 Aug 1.
Published in final edited form as: Dev Cell. 2009 Aug;17(2):175–186. doi: 10.1016/j.devcel.2009.06.017

Figure 7. Integrin-α9-EIIIA interaction regulates FN fibril assembly in primary human lymphatic endothelial cells.

Figure 7

(A) FN fibrils in primary human lymphatic endothelial cells (LECs). Integrin-α9-EIIIA interaction was blocked using antibodies against EIIIA (IST-9) or integrin-α9β1 (Y9A2), or siRNA against integrin-α9 or -α5, and stained with EIIIA antibodies.

(B) Quantification of FN fibrillogenesis in the LECs, in which integrin-α9-EIIIA interaction (IST-9, Y9A2, α9 siRNA) or integrin-α5/RGD-dependent integrin interactions (RGDSP peptide, α5 siRNA) were inhibited, in comparison to the control cells (untreated, ctrl siRNA or RGESP peptide). Data represent mean FN-EIIIA fiber length per cell (± s.d) from five randomly chosen view fields in two independent experiments. *** p< 0.003, n.s.= non-significant, p = 0.881 (Student T-test).

(C) qPCR of ITGA9 and FN-EIIIA in human LECs. Data represent mean ± s.d. of triplicates.

(D) siRNA mediated knock-down of integrin expression in primary human LECs. Western blot analysis of immunoprecipitated (IP) cell lysates using integrin-α9 or -α5 antibodies (upper panels). For the loading control, the total cell lysates (TCL) were blotted against α-tubulin and EIIIA.

(E) Conversion of DOC-soluble FN fibrils into insoluble stable matrix. DOC-insoluble (upper panel) and -soluble matrix (lower panel) isolated from the LECs were separated in non-reducing SDS-PAGE and probed for EIIIA.

(F) Immunofluorescent staining of wild type E18 mesenteric vessels using antibodies against integrin-α9 (left panel) and integrin-α5 (right panel). Note low levels of integrin-α5 expression in the valve (arrows) in comparison to strong staining in the blood vessel endothelia (arrowhead).

Scale bar = 20 μm.