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

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

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

Elastic Properties, Defect Thermodynamics, Electrochemical Window, Phase Stability, and Li⁺ Mobility of Li₃PS₄: Insights from First-Principles Calculations

The improved ionic conductivity (1.64 × 10^–4 S cm^–1 at room temperature) and excellent electrochemical stability of nanoporous β<i>-</i>Li₃PS₄ make it one of the promising candidates for rechargeable all-solid-state lithium-ion battery electrolytes. Here, elastic properties, defect thermodynamics, phase diagram, and Li⁺ migration mechanism of Li₃PS₄ (both γ and β phases) are examined via the first-principles calculations. Results indicate that both γ- and β-Li₃PS₄ phases are ductile while γ<i>-</i>Li₃PS₄ is harder under volume change and shear stress than β<i>-</i>Li₃PS₄. The electrochemical window of Li₃PS₄ ranges from 0.6 to 3.7 V, and thus the experimentally excellent stability (>5 V) is proposed due to the passivation phenomenon. The dominant diffusion carrier type in Li₃PS₄ is identified over its electrochemical window. In γ-Li₃PS₄ the <i>direct-hopping</i> of Li_i⁺ along the [001] is energetically more favorable than other diffusion processes, whereas in β<i>-</i>Li₃PS₄ the <i>knock-off</i> diffusion of Li_i⁺ along the [010] has the lowest migration barrier. The ionic conductivity is evaluated from the concentration and the mobility calculations using the Nernst–Einstein relationship and compared with the available experimental results. According to our calculated results, the Li⁺ prefers to transport along the [010] direction. It is suggested that the enhanced ionic conductivity in nanostructured β<i>-</i>Li₃PS₄ is due to the larger possibility of contiguous (010) planes provided by larger nanoporous β-Li₃PS₄ particles. By a series of motivated and closely linked calculations, we try to provide a portable method, by which researchers could gain insights into the physicochemical properties of solid electrolyte

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

PKG II inhibits EGF-induced Ras activation.

<p>The “pull-down” method was used to detect the activated Ras. Cell lysate was prepared and equal amounts of protein were incubated with GST-RBD beads as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061674#s4" target="_blank">materials and methods</a>. Binding complexes were collected by centrifugation, resolved by SDS-PAGE, transferred onto PVDF membrane and probed with anti-pan Ras antibody. The results shown are representative of three independent experiments.</p

PKG II blocks the phosphorylation of ERK.

<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 the phosphorylation of ERK. 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; ^&P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group).</p

PKG II prevents EGF-induced Tyr 992 phosphorylation of EGFR.

<p>AGS 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, cells were incubated with EGF (100 ng/ml) for 5 min. In Ad-LacZ+cGMP+EGF and Ad-PKGII+cGMP+EGF groups, cells were treated with 8-pCPT-cGMP for 1 h and then with EGF (100 ng/ml) for 5 min. Cells were harvested and lysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061674#s4" target="_blank">material and methods</a>. The cell lysate was subjected to Western blotting with antibody against Tyr 992 phospho-EGFR and EGFR. Total EGFR protein levels were used as loading control. 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