Immunofluorescence staining of p19 in human gastric mucosa.

<p>Immunofluorescence microscopic images depicting localization of p19 using a polyclonal anti-p19 antibody (red) in paraffin-embedded sections from antrum (A) and corpus (B) of <i>H. pylori</i> negative gastritis case. Both A and B are stitched overview images, a magnified region of interest (ROI) is indicated with a white square. Images (C-E) are the magnified ROI in A, whereas images (I-K) are the magnified ROI in B. Notably, gastric epithelial p19 expression showed two distinct staining patterns: membranous and cytoplasmic. The membranous was more clearly seen in the gastric superficial foveolar epithelium; the main site of host-bacteria interaction (J), whereas cytoplasmic pattern was mainly seen in the glandular compartment (D). As a function of fluorescence intensity, cells with stronger staining intensity were identified in the glandular neck zone of the antrum mucosa (A; arrowheads). Adjacent serial sections were used as negative controls to prove the specificity of the anti-p19 staining (F-H; L-N). Nuclei were stained with DAPI (blue). Scale bar (A-B)=250µm; (C-N)=50µm.</p

Boxplots of raw Cqs of the detected transcripts by RT-qPCR in non-infected AGS and MKN-28.

<p>Each box represents the lower quartile, the median, and the upper quartile score (vertical lines of the box). Means (small white squares) and outliers are also shown (black dots). Expression levels of detected transcripts were ordered in AGS cell line from highest (<i>ACTB</i>) to lowest (<i>IL23R</i>) based on their median values. Similar ranking order was seen in MKN-28 cells except for the least four abundant transcripts: <i>IL23A</i>, <i>IL12A</i>, <i>EBI3</i>, and <i>IL23R</i>.</p

Detection of IL-1β-induced nuclear translocation of NF-κB by immunofluorescence in C3842 cells (A–D) and SW1353 cells (E-H).

<p>In untreated cells and cells treated with Curcumin or Curcumin+IL-1β, NF-κB was detected in the cytoplasm, not in the nucleus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099296#pone-0099296-g006" target="_blank">Figure 6</a>). In cells treated with IL-1β nuclear translocation of NF-κB was evident.</p

Effect of Curcumin on VEGF-A expression.

<p>IL-1β-induced VEGF-A expression is blocked in C3842 and SW1353 chondrosarcoma cells after incubation with Curcumin.</p

VEGF-A protein expression in C3842 chondrosarcoma cells after treatment with IL-1β (10 ng/ml) for 1 to 24 h, in untreated cells (lane 2) and in VEGF-A overexpressing N109 renal carcinoma cells (lane 1), which served as positive control (P).

<p>β-actin is used as loading control.</p

Barcharts of <i>IL23A</i> median fold change by RT-qPCR in AGS and MKN-28 (n=4).

<p>BCM-300, P1, M1, and M2 are <i>H. pylori</i> strains. Both BCM-300 and P1 (wild-type) are CagA strains, whereas M1 and M2 are P1-derived isogenic mutants lacking CagA and VirB7, respectively. Generally, higher expression levels were seen in AGS more than MKN-28 cells. Significant upregulation of <i>IL23A</i> was seen in both cells with BCM-300, P1 (the highest), and M1 strain (P<.05), whereas no alteration was seen after M2 infection (non-functional T4SS) similar to that of the non-infected control samples (CO).</p

Correlation of <i>IL23A</i> with <i>IL8</i> and <i>EBI3</i> after infection with different <i>H. pylori</i> strains.

<p>Scatterplots of log-transformed fold changes of transcripts by RT-qPCR illustrate the relationship of <i>IL23A</i> with both <i>IL8</i> (panel A) and <i>EBI3</i> (panel B) transcripts after infection with different <i>H. pylori</i> strains using pair-wise Spearmen’s <i>rho</i> correlations. Robust regression line using an M estimator was plotted as well (red line). BCM-300, P1, M1, and M2 are <i>H. pylori</i> strains. Both BCM-300 and P1 (wild-type) are CagA strains, whereas M1 and M2 are P1-derived isogenic mutants lacking CagA and VirB7, respectively. BCM-300+Fi is infection with <i>H. pylori</i> BCM-300 bacteria after application of 0.22µm filter cap to prevent any physical contact with the cells, while permitting their secretory factors to pass through. Asterisks represent the significance levels (*P<.05, **<.01, ***<.001).</p

The time of the incubation with Curcumin prior to IL-1β treatment is essential for the inhibitory effect of Curcumin in chondrosarcoma cells.

<p>After an incubation of at least 60-1β treatment, Curcumin blocks phosphorylation of IκBα in C3842 chondrosarcoma cells.</p

An appropriate concentration of Curcumin is necessary for the inhibitory effect on IL-1 signaling.

<p>At least a Curcumin concentration of 15 µmol/l is required to block IκBα phosphorylation in C3842 (A) and SW1353 (B) chondrosarcoma cells.</p

Schematic diagram of IL-12 family.

<p>The diagram illustrates the currently known 5 chains (the innermost zone), composite cytokines with their proposed functions (the middle zone), and their receptors (the outermost zone). The five color-coded chains are either α chains (p19, p35, and p28) or β chains (p40 and Ebi3). Each functional cytokine is made of one α and one β chain. IL-12 (p35/p40) is a proinflammatory Th1 activator and stabilizer. Similarly, IL-23 (p19/p40) is a proinflammatory Th1 activator and Th17 stabilizer. On the other hand, IL-27 (p28/Ebi3) is an immunoregulatory cytokine mainly with an anti-inflammatory capacity. IL-35 (p35/Ebi3) is again anti-inflammatory and secreted by Treg cells. Therefore, the four cytokines with their divergent functions can promote a representative model of the immunological spectrum ranging from inflammation to tolerance. The unknown cytokine is a theoretical one and symbolizes the possibility of unraveling other unknown family members in the future. Each chain (p19, p40, p35, Ebi3, p28) binds to its specific receptor (IL23R, IL12RB1, IL12RB2, IL6ST, and IL27RA, respectively) and is coded with the same color of the corresponding chain (receptor names are not shown). Typically, a composite cytokine would require a β chain receptor for high affinity binding and an α chain receptor for signal transduction. For alternative terminology of IL-12 family members refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075192#pone-0075192-t001" target="_blank">Table 1</a>.</p