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

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

Additional file 1: of Cross-linked hyaluronan gel inhibits the growth and metastasis of ovarian carcinoma

Figure S1. The expression of EGFR in A2780 and SKVO3 cells. The celluar lysates were subjected to Western blotting with antibody against EGFR. Expression of β-actin was used at the same time as loading control. (TIFF 676 kb

Stereoselective Determination of Tebuconazole in Water and Zebrafish by Supercritical Fluid Chromatography Tandem Mass Spectrometry

A simple and sensitive method for the enantioselective determination of tebuconazole enantiomers in water and zebrafish has been established using supercritical fluid chromatography (SFC)-MS/MS. The effects of the chiral stationary phases, mobile phase, auto back pressure regulator (ABPR) pressure, column temperature, flow rate of the mobile phase, and compensation pump solvent were evaluated. Finally, the optimal SFC-MS/MS working conditions were determined to include a CO₂/MeOH mobile phase (87:13, v/v), 2.0 mL/min flow rate, 2200 psi ABPR, and 30 °C column temperature using a Chiralpak IA-3 chiral column under electrospray ionization positive mode. The modified QuEChERS method was applied to water and zebrafish samples. The mean recoveries for the tebuconazole enantiomers were 79.8–108.4% with RSDs ≤ 7.0% in both matrices. The LOQs ranged from 0.24 to 1.20 μg/kg. The developed analytical method was further validated by application to the analysis of authentic samples

PKG II prevents EGF-induced Tyr 1068 phosphorylation of EGFR.

<p>AGS cells were treated same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061674#pone-0061674-g002" target="_blank">Figure 2</a>. Western blotting was applied to detect Tyr 1068 phosphorylation of EGFR and densitometry analysis was performed to quantify the positive bands. A: A representative of initial results of three independent experiments. B: Results of densitometry analysis. The data shown are the means ± SD from 3 independent experiments (*P<0.05, compared to LacZ group and PKG II group; ^&P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group).</p

U73122/U1026/PKG II inhibits EGF-induced Cell migration.

<p>Migration activity of AGS cells was analyzed with transwell system. The cells were infected with Ad-LacZ or Ad-PKG II for 48 h and serum starved o/n. In Ad-LacZ+EGF and Ad-PKGII+EGF groups, EGF (100 ng/ml) was added to the culture medium; In Ad-LacZ+cGMP+EGF and Ad-PKGII+cGMP+EGF groups, cells were treated with 8-pCPT-cGMP for 1 h and then EGF (100 ng/ml) was added to the culture medium. The migration time was 12 h. A: Representative figures of migrated-cells stained by Giemsa (×200); B: The number of migrated cells in each group. The data shown are the means ± SD from 5 independent experiments, each performed in duplicate (*P<0.01, compared to LacZ group; ^&P<0.01, compared to LacZ+ EGF group).</p