Identification of the interaction between RhoC and IQGAP1.

<p>(<b>A</b>) BGC-823 cells growing on 100 mm plates were transiently co-infected with Ad-IQGAP1 and Ad-RhoC-V14, or Ad-IQGAP1-C and Ad-RhoC-V14 for 48 h. The cells were lysed and equal amounts of lysate protein were immunoprecipitated (IP) with anti-RhoC, anti-IQGAP1 antibodies or isotype-matched IgG. Whole cell lysate was used as a protein input control. (<b>B</b>) BGC-823 cells were transfected with above adenovirus for 24–48 h, and the co-localization of RhoC and IQGAP1 in cells were determined by Immunofluorescence microscopy using anti-RhoC and anti-IQGAP1 antibodies. Nuclei were stained by Hoechst 33342 (blue). (<b>C</b>) COS-7 cells were transiently co-infected with Ad-IQGAP1-C/Ad-IQGAP1 and Ad-RhoC-V14 for 48 h. The cells were undergoing the same Co-IP procedure described above. (<b>D</b>) COS-7 cells were transfected with above adenoviral vectors for 24–48 h, and the co-localization of RhoC and IQGAP1 in cells were shown by Immunofluorescence. The data are representative from three independent experiments with similar results.</p

The proliferation-stimulating effect of RhoC was blocked by IQGAP1 siRNA.

<p>(<b>A</b>) The protein expression levels of IQGAP1, IQGAP1-C and RhoC in BGC-823 cells. BGC-823 cells were transiently transfected with IQGAP1 siRNA or RhoC siRNA for 24 h. The transfected cells were afterwards infected with Ad-RhoC-V14, Ad-IQGAP1-C or Ad-IQGAP1 for additional 48 h followed by Western blotting. (<b>B</b>) RhoC depletion did not significantly affect IQGAP1 or IQGAP1-C induced proliferation of BGC-823 cells. (<b>C</b>) The silencing of IQGAP1 by siRNA markedly inhibited the RhoC-induced cell proliferation in BGC-823 cells. (MTT assay, *P<0.05; **P<0.01). The data are the means ± SD from three independent experiments each performed in duplicate.</p

The effect of IQGAP1 on proliferation of BGC-823 cells.

<p>(<b>A</b>) Western blot analysis of IQGAP1 expression in gastric cancer cell BGC-823 lines infected with Ad-LacZ, Ad-IQGAP1-C, Ad-IQGAP1-N or Ad-IQGAP1. (<b>B</b>) In the MTT assay, IQGAP1-C and IQGAP1 over expression cells both have more proliferation activity than control group (*P<0.05, compared to Ad-LacZ group). (<b>C</b>) The protein expression level of IQGAP1 in gastric cancer cell line BGC-823 transfected with IQGAP1 siRNA. (<b>D</b>) The proliferation of BGC-823 cells transfected with IQGAP1 siRNA were examined by MTT assay. (*P<0.05, compared to Control siRNA group). (<b>E</b>) BGC-823 cells were transiently transfected with plasmids Flag-IQGAP1, Flag-IQGAP1-C, or Flag-IQGAP1-N for 48 h. Western blotting showed the expression of IQGAP1, IQGAP1-N, and IQGAP1-C constructs in BGC-823 cell lines. Equal amounts of cell lysate from each group were loaded and blotted with anti-IQGAP1 antibodies (against C-terminal fragment or N- terminal fragment). (<b>F</b>) BrdU assay was used for analysis cell proliferation. Representative images of BGC-823 cells expressing the indicated IQGAP1 constructs were stained with antibodies against BrdU (second panels red) and Hoechst 33342 for nuclei (first panel, blue). The percentage of cells with BrdU incorporation was calculated. The mean ± SD of three independent experiments is presented (*P<0.05).</p

The schematic diagram of crosstalk between RhoC and IQGAP1 in gastric cancer.

<p>When RhoC is stimulated by extracelluar or intracelluar signals, it binds with the scaffold protein IQGAP1, influences the expression of cell cycle-related proteins such as cyclin D1 and cyclin B, affects G1-S transitions in the cell cycle, and then causes the change in cell proliferation. The signal transduction event through which IQGAP1 affects the expression of cyclin still needs to be elucidated.</p

The effects of RhoC and IQGAP1 on expression of cell cycle-related proteins.

<p>(<b>A</b>) BGC-823 cells were infected with Ad-IQGAP1, Ad-IQGAP1-C or Ad-RhoC-V14 for 48 h, and Western blot was used to analyze the expressions of cyclin E, cyclin D1 cyclin B and CDK. (<b>B</b>) BGC-823 cells were transfected with IQGAP1 siRNA or RhoC siRNA for 72 h, and the expressions of cyclin E, cyclin D1, cyclin B and CDK were analyzed by Western blotting. (<b>C</b>) The protein expressions of cyclin E, cyclin D1, cyclin B and CDK in BGC-823 cells which were transiently transfected with IQGAP1 siRNA or RhoC siRNA for 24 h and afterwards infected with Ad-RhoC-V14, Ad-IQGAP1-C or Ad-IQGAP1 for additional 48 h (Results of Western blotting).</p

Additional file 2: Table S2. of Circular RNAs: emerging cancer biomarkers and targets

The potential of circRNAs as cancer biomarkers. (DOCX 32 kb

Protein expression confirmation by western blot.

<p>(A) Left panel: Results of Western blot analysis performed with specific protein antibodies and protein extracts from mouse skin. (B) Right panel: Magnified spot images of the same molecular weights distributed in 2-DE gels.</p

Potential biological network of differentially expressed proteins during hair cycling.

<p>Cellular processes (yellow squares) involved in each cluster and all relevant proteins (red ovals) validated by literates (the line). Regulation events are displayed with arrows. (A) The cellular processes of high expressed proteins in telogen involved in C1; (B) The cellular processes of high expressed proteins in anagen involved in C2; (C) The cellular processes of high expressed proteins in catagen involved in C3.</p

Cluster analysis of the expression levels of 44 differentially expressed protein spots in the three phases.

<p>Three clusters (C1, C2, C3) corresponding to three distinct expression patterns were identified. Green represents down-regulated expression, whereas red indicates up-regulated levels.</p

The GO enrichment analysis performed by the DAVID bioinformatics resources online.

<p>The results were grouped based on the biological process (BP), molecular function (MF) and cellular component (CC). Statistically significant differences (p-value <0.05) were determined using Fisher’s exact test.</p