The correlation among the selected proteins.

<p>The GRP78 in these 12 pairs of tissues was expressed correlatively with that of ApoAI (r2: −0.61) and A1AT (r2: −0.49) individually. Meanwhile, ApoAI was expressed correlatively with that of A1AT (r2: 0.62). However, GSTpi and GKN-1 seemed to be independent. **p<0.01. *p<0.05.</p

Immunohistochemistric stains were used to identify the location of the putative proteins.

<p>The up-regulated proteins, GRP78 and GSTpi, were specifically located on the gastric adenocarcinoma cells. Contrarily, the down-regulated proteins including ApoAI and A1AT were present on the normal gastric glands. Another down-regulated protein, GKN-1, was shown to appear on the mucosa of gastric tissue.</p

The detailed comparison between normal and tumorous tissue from 2D-DIGE.

<p>The selected putative proteins were presented as stereopictures and enlarged scales gel images. These proteins were selected according to the significant difference below 0.05 (<i>p</i><0.05). In addition, the duplicated identification of ATPB, PDIRP5, ApoAI and GSTpi on adjacent spots indicated that they had a different modification.</p

Discovery of Tumor Markers for Gastric Cancer by Proteomics - Figure 3

<p>(A) By Western blotting, 12 paired of gastric cancer and normal tissues was used to validate the putative proteins including GRP78, GSTpi, A1AT, ApoAI and GKN-1. (B) The GSTpi and GRP78 were significantly over-expressed in gastric cancer tissues.Down-expressions of A1AT, ApoAI and GKN-1 were noted. **<i>p</i><0.01. *<i>p</i><0.05.</p

The 2D-DIGE results paired tissue samples.

<p>The differential proteins presented on gels. (left): normal; (right): cancer. Thirty-six proteins were picked by DeCyder statistical software, but only fifteen proteins were identified by PMF or PFF technique. The gel conditions: pI: 4–7; gel: 4–12%.The analytical ranges were from pI 4 to 7 forisoelectric focusing and from 4% to 12% gradient gel for sodium dodecyl sulfate- polyacrylamide gelelectrophoresis.</p

The correlations between clinical stages of gastric cancer and the expressions of proteins.

<p>Although GRP78 and GSTpi were increased in different stages of gastric cancer, no statistical significance could be found. A trend of increase with clinical stage was found in GRP78.</p

Gastric myofibroblast express CD90, α-smooth muscle actin, and class II MHC.

<p>Gastric myofibroblast express <b>A</b>) CD90 by flow cytometry compared to the solid peak isotype control. These cells also express <b>B</b>) α-smooth actin and class II MHC, where class II MHC is upregulated by exposure to <i>H. pylori</i> as shown by confocal microscopy. Class II MHC is also upregulated by <b>C</b>) incubation with CD4⁺ T cells and <b>D</b>) <i>H. pylori</i> exposure by flow cytometry. Figures are representative of 3 experiments in duplicate.</p

RORγt expressing cells proliferate in culture with GMF in a class II MHC dependent manner.

<p>CD4⁺ T cells labeled with CFSE and gated on RORγt⁺ cells for representative histograms for <b>A</b>) non-proliferating control, <b>B</b>) CD4⁺ T cells in culture with normal GMF <b>C</b>) CD4⁺ T cells in culture with <i>H. pylori-</i>exposed GMF, <b>D</b>) CD4⁺ T cells in culture with <i>H. pylori-</i>exposed GMF pre-incubated with class II MHC blocking antibodies <b>E</b>) CD4⁺ T cells in culture with cancer GMF <b>F</b>) CD4⁺ T cells in culture with cancer GMF pre-incubated with class II MHC blocking antibodies. <b>G</b>) Compiled data from 3 experiments in triplicate show the percent proliferating cells from CD4⁺ T cells in culture with normal GMF, <i>H. pylori-</i>exposed GMF, and cancer GMF with class II MHC blocking. (n = 9). <b>*</b><i>p</i><0.05 <i>H. pylori</i> treated or cancer and class II MHC blockade.</p

Th17 development increases in culture with <i>H. pylori-</i>exposed and cancer gastric myofibrobroblasts.

<p><b>A</b>) N-GMF and Hp-GMF primed CD4⁺ T cells were analyzed for RORγt expression by flow cytometry. <b>B</b>) N-GMF and C-GMF primed CD4⁺ T cells were analyzed for RORγt expression by flow cytometry. <b>C</b>) Compiled data for all experiments of N-GMF, Hp-GMF, and C-GMF primed CD4⁺ T cells analyzed for RORγt expression by flow cytometry. <b>D</b>) IL-17A levels were measured in co-cultures of CD4⁺ T cells with GMF by Luminex bead array. The RNA levels from GMF primed CD4⁺ T cells were also analyzed by quantitative real time PCR for <b>E</b>) RORγ and IL-17A. The mRNA levels were normalized to 18S. Data represent mean of mRNA fold increase ± standard errors from three duplicate experiments from 3 sets of GMF (n = 18). <b>*</b><i>p</i><0.05, between CD4⁺ and CD4⁺ incubated with GMF, <b>**</b><i>p</i><0.05 between untreated and <i>H. pylori</i> treated or cancer.</p

Th17 are increased in human tumor and <i>H. pylori</i>-infected tissues samples.

<p>Quantitative real time RT-PCR data indicates <i>H. pylori</i> infected tissues compared to unmatched normal tissues have increases in <b>A</b>) RORγ and <b>B</b>) IL-17A mRNA levels. Human gastric tumor samples compared to matched normal samples also have increased <b>C</b>) RORγ and <b>D</b>) IL-17A. The mRNA levels were normalized to 18S. Data represent mean of mRNA fold increase ± standard errors from triplicates compared to the levels in normal samples. * <i>p</i><0.05.</p