STAT1 knockdown blocks IFNγ-induced IL-6 and IL-8 in monolayer HEECs.

<p>STAT1 siRNA and non-specific control siRNA (negative siRNA) were transfected into monolayer HEECs 72 h before stimulation. (<i>A</i>) STAT1 expression was evaluated using RT-qPCR after transfection. (<i>B</i>) STAT1 production was evaluated by western blot analysis after transfection. (<i>C</i>, <i>D</i>) The production of IL-6 (<i>C</i>) and IL-8 (<i>D</i>) 24 h after IFNγ (30 ng/ml) stimulation was detected by ELISA in the supernatants of negative siRNA and IL-33 siRNA treated groups. Each value represents the mean ± SD of 3 independent experiments. **<i>P</i> < 0.01.</p

IFNγ-induced cytokine production is p38 MAPK and JAK/STAT1 dependent.

<p>ALI-cultured HEECs were pre-incubated with inhibitors of JAKs (2 μM, JAKi), p38 MAPK (40 μM, SB), PKA (10 μM, H89), or STAT1 (20 μM, EGCG) for 1 h, and subsequently co-incubated with IFNγ (30 ng/ml). (<i>A</i>) Cells were harvested 10 h after IFNγ stimulation to evaluate IL-33 production by western blot analysis (loading control: β-actin). (<i>B</i>) Media from the basal compartment were harvested 24 h after IFNγ stimulation to analyze IL-6, IL-8, RANTES, and MCP-1 production using the Bio-Plex assay. Each value represents the mean ± SD of 3 independent experiments. **<i>P</i> < 0.01 vs. Control. ^## <i>P</i> < 0.01 vs. IFNγ group.</p

IFNγ, but not TNF-α, upregulates nuclear IL-33.

<p>(<i>A</i>, <i>B</i>) IL-33 mRNA was analyzed by RT-qPCR. (<i>A</i>) ALI-cultured HEECs were stimulated with IFNγ (30 ng/ml), TNF-α (20 ng/ml), or both from the basal compartment, and harvested after the indicated time points. (<i>B</i>) ALI-cultured HEECs were stimulated with IFNγ (0.1, 1, 5, and 30 ng/ml) and harvested after 6 h. Each value represents the mean ± SD of 3 independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01. (<i>C</i>) Immunofluorescence staining of IL-33 (red) in ALI-cultured HEECs after IFNγ (30 ng/ml) stimulation at the indicated time points was performed. DAPI (blue) was used as the nuclear marker. Bar = 50 μm. (<i>D</i>) Relative levels of IL-33 in total or nuclear protein extract were assessed by western blot analysis at the indicated time points after IFNγ (30 ng/ml) stimulation.</p

Exogenous IL-33 does not induce cytokines.

<p>(<i>A)</i> Relative levels of phosphorylated NF-κB p65 (p-p65) were assessed by western blot analysis after IL-33 (50 ng/ml, 1 h) stimulation. β-actin was used as a loading control. (<i>B</i>, <i>C</i>) ALI-cultured HEECs were stimulated with IL-33 from the basal compartment (10, 100 ng/ml) for 24 h. IL-8 and IL-6 production was determined by ELISA. Each value represents the mean ± SD of 3 independent experiments.</p

Colocalization of IL-33 and pan-cytokeratin in ALI-cultured HEECs.

<p>Immunofluorescence staining of IL-33 (red) and pan-cytokeratin (green) in ALI-cultured HEECs after IFNγ (30 ng/ml, 6 h) stimulation was performed. DAPI (blue) was used as the nuclear marker. Bar = 50 μm.</p

Organocatalytic Asymmetric Mannich Reaction of 3‑Hydroxyoxindoles/3-Aminooxindoles with in Situ Generated <i>N</i>‑Boc-Protected Aldimines for the Synthesis of Vicinal Oxindole–Diamines/Amino Alcohols

A highly efficient asymmetric Mannich reaction of 3-monosubstituted 3-aminooxindoles/3-hydroxyoxindoles with in situ generated <i>N</i>-Boc-protected aldimines catalyzed by the chiral bifunctional thiourea–tertiary amine catalyst has been developed. Under mild reaction conditions, a series of structurally diverse vicinal oxindole–diamines/amino alcohols were smoothly obtained in moderate to high yields (up to 99%) with good to excellent diastereoselectivities and enantioselectivities (up to 95:5 dr and 96% ee). The synthetic application of this protocol was also demonstrated by the versatile transformation of chiral vicinal oxindole–diamine<b>/</b>amino alcohol into spirocyclic oxindoles

Claudin-18 knockdown by siRNA. (A) Claudin-18 siRNA significantly decreases the mRNA level of claudin-18 in MKN74.

<p>(B) Staining of claudin-18 is reduced by claudin-18 siRNA treatment. Bar = 50 µm. Each value represents the mean ± SD of 3 independent experiments. **<i>P</i><0.01 vs. control or control siRNA.</p

HE staining and immunofluorescence staining of claudin-18 in gastric cancer (GC) lesions and surrounding gastric mucosa.

<p>(A) HE staining of gastric mucosa with non-intestinal metaplasia (non-IM), intestinal metaplasia (IM), and cancer. Claudin-18 is detected in gastric mucosa with non-IM at the surface border and lateral membrane of the epithelial cells, but not with IM or cancer. White bar = 100 µm, Yellow bar = 50 µm. (B) Claudin-18 level is significantly lower in GC than in non-IM or IM. The level is also significantly lower in IM than in non-IM. ^#<i>P</i><0.05 vs. IM. **<i>P</i><0.01 vs. non-IM.</p

Clinicopathological features of gastric cancer patients.

<p>T1a; tumor confined to the mucosa, T1b; tumor confined to the submucosa,</p><p>U; upper third, M; middle third, L; lower third <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074757#pone.0074757-JapaneseGastricCancerAssocciation1" target="_blank">[18]</a></p

Effect of claudin-18 knockdown on the proliferation and invasion of GC cells.

<p>(A) Claudin-18 siRNA treatment significantly induces cell proliferation. (B) Claudin-18 siRNA-transfected MKN74 cells show a significantly higher invasion ratio compared to control or control siRNA-transfected cells. Typical images are presented at the top. Bar = 500 µm. Each value represents the mean ± SD of 3 independent experiments. **<i>P</i><0.01 vs. control or control siRNA.</p