Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dynamics of the IL-33/ST2 network in the progression of human colorectal adenoma to sporadic colorectal cancer

doi: 10.1007/s00262-014-1624-x

Figure Lengend Snippet: Dynamic changes in IL-33 and ST2 transcripts along the colorectal adenoma–carcinoma sequence. The quantitative real-time PCR results showed that the expression of IL-33 mRNA was significantly increased to a ~ninefold higher level in adenoma tissues (gray bar in a); it was also higher in sporadic CRC tissues (black bar in a) than in normal controls (white bar in a), but significantly lower compared with the adenomas. The expression of ST2 mRNA in adenoma tissues was increased to a ~threefold higher level and slightly increased in CRC tissues compared with normal controls (b). (Y axes in a, b are fold changes relative to normal controls; P values are derived from the Mann–Whitney test.)

Article Snippet: The following primary antibodies were used: goat antihuman IL-33 polyclonal antibody (working dilution 1:100; R&D systems, Minneapolis, MN, USA) and mouse antihuman ST2 monoclonal antibody (working dilution 1:100; Medical & Biological Laboratories Co. Ltd., Nagoya, Japan).

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, MANN-WHITNEY

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dynamics of the IL-33/ST2 network in the progression of human colorectal adenoma to sporadic colorectal cancer

doi: 10.1007/s00262-014-1624-x

Figure Lengend Snippet: Kaplan–Meier curves of overall survival differences among patients with sporadic CRC. The Kaplan–Meier analysis shows that the expression levels of IL-33 (a) and ST2 (b) do not predicate the overall survival time in patients with sporadic CRC (both P values determined by the log-rank test)

Article Snippet: The following primary antibodies were used: goat antihuman IL-33 polyclonal antibody (working dilution 1:100; R&D systems, Minneapolis, MN, USA) and mouse antihuman ST2 monoclonal antibody (working dilution 1:100; Medical & Biological Laboratories Co. Ltd., Nagoya, Japan).

Techniques: Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dynamics of the IL-33/ST2 network in the progression of human colorectal adenoma to sporadic colorectal cancer

doi: 10.1007/s00262-014-1624-x

Figure Lengend Snippet: Photograph presentations of IL-33 and its receptor, ST2, in the tumor stroma and the adenomatous/cancerous epithelium. Immunohistochemical (IHC) results show that IL-33 immunoreactivity was not observed in the normal epithelium, and a low density of IL-33-positive cells was found in the lamina propria in the control (a). In both the adenoma and CRC, IL-33 immunoreactivity was frequently observed in tumor-associated microvessels (arrow in b, c) and adenomatous/cancerous epithelium (arrow in b for adenoma and inserted image in 3C for CRC). ST2 immunoreactivity was observed in both the epithelium (arrow head) and microvessels (arrow) in all three groups (d for control, e for adenoma and f for CRC). (a–e IHC, counterstained with hematoxylin, original magnification 200×.)

Article Snippet: The following primary antibodies were used: goat antihuman IL-33 polyclonal antibody (working dilution 1:100; R&D systems, Minneapolis, MN, USA) and mouse antihuman ST2 monoclonal antibody (working dilution 1:100; Medical & Biological Laboratories Co. Ltd., Nagoya, Japan).

Techniques: Immunohistochemical staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dynamics of the IL-33/ST2 network in the progression of human colorectal adenoma to sporadic colorectal cancer

doi: 10.1007/s00262-014-1624-x

Figure Lengend Snippet: Density grading scores of IL-33- and ST2-positive cells in adenomas and CRC tissues. The semi-quantitative results showed that the density of IL-33-positive cells in the adenomatous epithelium (gray bar in a) was higher than in the controls. The density of IL-33-positive cells was also higher in the CRC epithelium (black bar in a) compared with the controls, but lower than the adenomatous epithelium (Fig. a). The density of IL-33-positive stromal cells was increased in the adenoma stroma, with a smaller increase in magnitude of density in the CRC stroma (b). The density of ST2-positive cells in the adenomatous epithelium was non-statistically increased and was unchanged in the CRC cancerous epithelium (c). The density of ST2-positive cells in the stroma showed a gradually increasing trend from the control to adenoma to CRC, but these differences were not statistically significant (d). To examine the expression of IL-33/ST2 in endothelial cells, the number of IL-33-positive tumors associated with microvessel density (MVD) and the number of ST2-positive tumors associated with MVD were counted. The results show that the IL-33-positive MVD was significantly increased in the adenoma stroma and non-statistically increased in the CRC tumor stroma compared with normal lamina propria (e). The number of ST2-positive microvessels was greatly increased in both the adenoma stroma and the CRC tumor stroma compared with the normal lamina propria (f). (HPF high-power field.)

Article Snippet: The following primary antibodies were used: goat antihuman IL-33 polyclonal antibody (working dilution 1:100; R&D systems, Minneapolis, MN, USA) and mouse antihuman ST2 monoclonal antibody (working dilution 1:100; Medical & Biological Laboratories Co. Ltd., Nagoya, Japan).

Techniques: Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dynamics of the IL-33/ST2 network in the progression of human colorectal adenoma to sporadic colorectal cancer

doi: 10.1007/s00262-014-1624-x

Figure Lengend Snippet: Phenotypic characterization of IL-33-expressing cells in the adenoma and CRC tumor stromas. Double IHC with IL-33/CD34 and IL-33/SMA-alpha revealed that an increase in IL-33-positive cells (red) in the adenoma and CRC tumor stromas was frequently co-localized with CD34-labeled microvessels (blue) (b, c) and SMA-alpha-labeled myofibroblasts (blue) (e, f) compared with the normal lamina propria (a, d). (a–f double IHC, original magnification 400×; counterstaining was not applied.)

Article Snippet: The following primary antibodies were used: goat antihuman IL-33 polyclonal antibody (working dilution 1:100; R&D systems, Minneapolis, MN, USA) and mouse antihuman ST2 monoclonal antibody (working dilution 1:100; Medical & Biological Laboratories Co. Ltd., Nagoya, Japan).

Techniques: Expressing, Labeling

Journal: JID Innovations

Article Title: Nuclear IL-33 Plays an Important Role in EGFR-Mediated Keratinocyte Migration by Regulating the Activation of Signal Transducer and Activator of Transcription 3 and NF-κB

doi: 10.1016/j.xjidi.2023.100205

Figure Lengend Snippet: HB-EGF at 30 ng/ml significantly stimulates IL-33 expression and accelerates KC wound closure. ( a ) KCs were treated with different concentrations of HB-EGF for 6 hours. IL33 mRNA expression was examined by real-time RT-PCR. The data represent the results of a single experiment with three samples per test point. ( b ) KCs were treated with different concentrations of HB-EGF for 8 hours. IL-33 protein expression was examined by western blotting. The relative IL-33 protein levels represent the ratio of IL-33 intensity in different HB-EGF−treated groups divided by that in the control group (0 ng/ml HB-EGF). The dot plot data represent the results obtained from three separate experiments. ( c ) A scrape-wound healing assay was performed in almost confluent cultures using an 8-mm scraper. The cultures were scraped, washed, and treated with different concentrations of HB-EGF. The pictures were taken 40 hours after scratching. Bar = 100 mm. All tests were repeated three times, and the results from the representative experiments are shown here. Data are shown as mean ± SD, with n = 3 for each test point. Significance was tested by one-way ANOVA (for a and b ). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 versus the control group (0 ng/ml HB-EGF). h, hour; HB-EGF, heparin-binding epidermal GF; KC, keratinocyte.

Article Snippet: Human IL33 gene open reading frame cDNA clone expression plasmid, c-Myc tag (pc-Myc−IL-33) was obtained from Sino Biological (Beijing, China, catalog number HG10368-CM).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Wound Healing Assay, Binding Assay

Journal: JID Innovations

Article Title: Nuclear IL-33 Plays an Important Role in EGFR-Mediated Keratinocyte Migration by Regulating the Activation of Signal Transducer and Activator of Transcription 3 and NF-κB

doi: 10.1016/j.xjidi.2023.100205

Figure Lengend Snippet: HB-EGF rapidly induces IL-33 expression but does not stimulate IL-33 secretion. ( a ) KCs were treated with 30 ng/ml HB-EGF for the indicated times. The levels of IL33 mRNA were decided by real-time RT-PCR. ( b ) The levels of IL-33 protein were detected by western blotting and calculated by NIH, and the relative IL-33 protein levels represent the ratio of IL-33 intensity in different HB-EGF−treated groups divided by that in the control group (HB-EGF at 0 h). ( c ) KCs were treated with or without HB-EGF (30 ng/ml) for 24 h, cell damage was performed by scratching a lot, and the supernatants were collected for the detection of IL-33 by ELISA. All tests were repeated three times, and the results from the representative experiments are shown here. The dot blot data represents the results of a single experiment with three samples per test point (for a and c ), or the dot plot data are the results of three individual experiments (for b) . Data are shown as mean ± SD, with n = 3 for each test point. Significance was tested by one-way ANOVA (for a and b ) or by a paired t -test (for c) . ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 versus the control group (HB-EGF at 0 h). # P < 0.05, ## P < 0.01, and ### P < 0.001 versus the indicated group. h, hour; HB-EGF, heparin-binding epidermal GF; KC, keratinocyte; NIH, National Institutes of Health; NS, not significant.

Article Snippet: Human IL33 gene open reading frame cDNA clone expression plasmid, c-Myc tag (pc-Myc−IL-33) was obtained from Sino Biological (Beijing, China, catalog number HG10368-CM).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Dot Blot, Binding Assay

Journal: JID Innovations

Article Title: Nuclear IL-33 Plays an Important Role in EGFR-Mediated Keratinocyte Migration by Regulating the Activation of Signal Transducer and Activator of Transcription 3 and NF-κB

doi: 10.1016/j.xjidi.2023.100205

Figure Lengend Snippet: HB-EGF rapidly induces IL-33 expression in KCs through EGFR-mediated activation of Ras/ERK and PI3K/Akt pathways. After KCs were treated with HB-EGF (30 ng/ml) for the indicated times, ( a ) the phosphorylation of EGFR, ERK, Akt, and STAT3 and ( b ) the effects of specific inhibitors (3 mM AG1478, 10 mM U0126, or 10 mM wortmannin) on EGFR, ERK, Akt, and STAT3 activation were examined by western blotting; the relative protein levels of p-EGFR, p-ERK, p-Akt, and p-STAT3 represent the ratio of specific phosphorylated protein intensity that was corrected already by its total protein intensity in different HB-EGF (+) groups divided by that in the control group (HB-EGF [−] group) (for b ). ( c ) KCs were pretreated with DMSO or specific inhibitors for 30 minutes and then stimulated with or without HB-EGF for 8 h, and IL33 mRNA expression was examined by real-time RT-PCR. ( d ) KCs were infected with Ax for 24 h and then stimulated with or without HB-EGF for the indicated times; the effects of infection with STAT3F-expressing Ax on STAT3 activation and IL33 mRNA expression were detected by western blotting and real-time RT-PCR, respectively. All tests were repeated three times, and the results from the representative experiments are shown here. The dot plot data indicate the results of three individual experiments ( for b) , or the dot blot data represent the results of a single experiment with three samples per test point (for c and d ). Data are shown as mean ± SD, with n = 3 for each test point. Significance was tested by one-way ANOVA (for b ) or two-way ANOVA (for c and d ). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 versus HB-EGF (+)/inhibitor (−) group (for b and c) or HB-EGF at 0 h/LacZ-expressing Ax group (for d ). # P < 0.05, ## P < 0.01, and ### P < 0.001 versus HB-EGF (+)/inhibitor (−) group (for b ) or the indicated group (for c ). Ax, adenovirus vector; Akt, protein kinase B; ERK, extracellular signal–regulated kinase; h, hour; HB-EGF, heparin-binding epidermal GF; KC, keratinocyte; NS, not significant; p-Akt, phosphorylated protein kinase B; p-EGFR, phosphorylated EGFR; p-ERK, phosphorylated extracellular signal–regulated kinase; PI3K, phosphoinositide 3-kinase; p-STAT3, phosphorylated signal transducer and activator of transcription 3; rhST, recombinant human ST; STAT3, signal transducer and activator of transcription 3.

Article Snippet: Human IL33 gene open reading frame cDNA clone expression plasmid, c-Myc tag (pc-Myc−IL-33) was obtained from Sino Biological (Beijing, China, catalog number HG10368-CM).

Techniques: Expressing, Activation Assay, Western Blot, Control, Quantitative RT-PCR, Infection, Dot Blot, Plasmid Preparation, Binding Assay, Recombinant

Journal: JID Innovations

Article Title: Nuclear IL-33 Plays an Important Role in EGFR-Mediated Keratinocyte Migration by Regulating the Activation of Signal Transducer and Activator of Transcription 3 and NF-κB

doi: 10.1016/j.xjidi.2023.100205

Figure Lengend Snippet: Nuclear IL-33 is required for HB-EGF/EGFR-mediated KC migration and wound closure. ( a ) A scratch wound healing assay was performed to evaluate the involvement of IL-33/ST2 signaling in KC migration and wound closure. After scratching wounds were made by a 6-mm scraper, the cultures were treated with HB-EGF (30 ng/ml) or mature IL-33 (100 ng/ml) together with or without recombinant human ST2/IL-33R Fc chimera protein (300 ng/ml) for 28 hours. The photos and the unrecovered wound area of several wounded spots (compared with that of the 0-hour group) calculated by ImageJ are presented here. Bar = 100 mm. ( b ) After transfection with control or IL-33 siRNA, KCs were treated with HB-EGF for 8 hours or 12 hours. IL33 mRNA and protein expressions were detected by real-time RT-PCR and western blotting, respectively; the relative levels of IL33 mRNA and protein are presented together with IL-33 protein bands. ( c ) After siRNA transfection, a scratch wound healing assay was performed by making wounds with a 6-mm scraper and adding HB-EGF or vehicle into cultures. The effect of IL-33 knockdown on wound closure was monitored, and the scraped area at the indicated times was quantified by ImageJ. Bar = 100 mm. ( d ) The effect of IL-33 knockdown on cell migration was quantified with a Boyden chamber migration assay. All tests were repeated three times, and the results from the representative experiments are shown here. The dot blot data represent the results of a single experiment with six (for a , c , and d ) or three (for b, left) samples per test point, or the dot plot data indicate the results of three individual experiments (for b , right ) . Data are shown as mean ± SD, with n = 6 (for a , c , and d ) or n = 3 (for b ) for each test point. Significance was tested by a paired t -test (for a , b, and c ) or two-way ANOVA (for d ). # P < 0.05 and ## P < 0.01 versus the indicated group. cr, control; h, hour; HB-EGF, heparin-binding epidermal GF; KC, keratinocyte; NS, not significant; siRNA, small interfering RNA.

Article Snippet: Human IL33 gene open reading frame cDNA clone expression plasmid, c-Myc tag (pc-Myc−IL-33) was obtained from Sino Biological (Beijing, China, catalog number HG10368-CM).

Techniques: Migration, Wound Healing Assay, Recombinant, Transfection, Control, Quantitative RT-PCR, Western Blot, Knockdown, Dot Blot, Binding Assay, Small Interfering RNA