Representative results of immunohistochemistry of Annexin A1, S100A9 and Vimentin in the NPC.

<p>Immunohistochemical staining of Annexin A1(A), S100A9(B) and Vimentin (C) in the NPC tissues. Original magnification, ×200. (D) Combined staining score (extension + intensity) distribution of Annexin A1, S100A9 and Vimentin in the 32 cases of NPC tissues.</p

In vitro migration and invasion assays and wound healing assays.

<p>(A) In vitro migration of the 5-8F cells, 6-10B cells and their transfectants (B) In vitro invasion of the 5-8F cells, 6-10B cells and their transfectants. All the cells were measured using Transwell chambers. Bar chart showing the average number of every kind of cells which penetrated the membrane correspondingly. Original magnification, x400. (C) Wound healing assays. Data and representative images for six cell lines, as shown in the figure. Bars represent the percentage of wound closure. Original magnification, x40. Three independent experiments were performed.</p

Confirmation of Annexin A1/S100A9/Vimentin interaction in 6-10B NPC cells by co-immunoprecipitation and Western blot analysis.

<p>(A) Annexin A1 can be detected in Annexin A1, S100A9 and Vimentin's co-immunoprecipitated complex by Westernblot analysis. (B) Vimentin can be detected in Annexin A1, S100A9 and Vimentin co-immunoprecipitated complex by Western blot analysis. (C) S100A9 can be detected in Annexin A1, S100A9 and Vimentin co-immunoprecipitated complex by Western blot analysis, but the three proteins cannot be detected in the non-immune Ig G control. Three independent experiments were performed.</p

Analysis of protein–protein interaction using String 9.1(http://string-db.org/newstring_cgi/show_input_page.pl).

<p>(A) Integrated distribution network of all the interactions of Annexin A1-associated proteins is showed. Nodes show the logogram of foregone Annexin A1-associated proteins and interactions marked by lines. (B) Partial enlarged detail of interactions of Annexin A1-associated proteins. Annexin A1/S100A9/Vimentin can form complexes in NPC cells.</p

Effects of Annexin A1 modulation on the Vimentin and S100A9 expression.

<p>Western blot analysis showing the expression levels of Annexin A1, Vimentin and S100A9 in 5-8F and 6-10B NPC cells and their transfectants, 5-8F: untransfected 5-8F cells; 5-8F-Annexin A1(+): transfected 5-8F cells with Annexin A1-expressing plasmid; 5-8F-Annexin A1(−): transfected 5-8F cells with control vector; 6-10B:untransfected 6-10B cells; 6-10B siRNA(+): transfected 6-10F cells with Annexin A1 siRNA plasmid; 6-10B-siRNA(−): transfected 6-10B cells with control vector. β-Actin was used as an internal control for loading.</p