Journal: Cell Communication and Signaling : CCS

Article Title: The IL-33-ST2 axis plays a vital role in endometriosis via promoting epithelial–mesenchymal transition by phosphorylating β-catenin

doi: 10.1186/s12964-024-01683-x

Figure Lengend Snippet: Expression Patterns and Inducing Factors of Interleukin-33 (IL-33) in Endometriotic milieu. A Levels of IL-33 were detected by ELISA in the peritoneal fluid of patients with EMs and controls. B Real-time PCR for analyzing the mRNA expression of IL-33 in primary endometrial stromal cells (ESCs). C Levels of IL-33 secretion in the cell supernatant of different primary ESCs. D - I : Changes of IL-33 expression in ESC after different treatments: mRNA ( D ) and protein ( E ) level of IL-33 after H 2 O 2 and/or NAC treatment; mRNA ( F ) and protein (G) levels of IL-33 after estradiol (E 2 ) treatment; mRNA ( H ) and protein ( I ) levels of IL-33 after TGF-β1 treatment. J Immunofluorescence co-localization of vimentin and ST2 (IL-33 receptor) in endometrial tissues. (blue:DAPI, green:vimentin, red: ST2) (Scale bars, 20 μm). K Immunofluorescence co-localization of vimentin and E-cadherin in endometrial tissues. (blue:DAPI, green:vimentin, red: E-cadherin) (Scale bars, 20 μm). Ctrl-EU, eutopic endometrium of controls; EMs-EU, eutopic endometrium of patients with endometriosis; EMs-EC, ectopic lesions. All data were analyzed using one-way ANOVA followed by Dunnett’s post-hoc test . Spearman’s correlation analysis was used to analyze the correlation between ST2 and vimentin expression in humans.* P < 0.05, ** P < 0.01, *** P < 0.001, data are presented as mean ± SEM

Article Snippet: The following IHC antibodies were used: anti-human ST2 (1:200), anti-human CCN4 (1:200), and anti-human E-cadherin (1:500) (abcam, Cambridge, UK); anti-human vimentin (1:100) (Cell Signaling Technology, Boston, USA); anti-human/mouse Fra-1 (1:200), anti-mouse CCN4 (1:100), and anti-mouse vimentin (1:100) (Affinity Biosciences, China); The primary antibodies used are listed in Supplementary Table 1.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: The IL-33-ST2 axis plays a vital role in endometriosis via promoting epithelial–mesenchymal transition by phosphorylating β-catenin

doi: 10.1186/s12964-024-01683-x

Figure Lengend Snippet: IL-33 is Crucial for the Epithelial–Mesenchymal Transition (EMT) Process in EECs. A RNA expression of EMT-related genes ( VIM, CDH1, CDH2, and CTNNB1 ) in EECs and 12Z cells. B EMT-related protein (vimentin, E-cadherin, N-cadherin, and β-catenin) were detected using western blot in EECs and 12Z cells. C mRNA expression of EMT-related genes ( VIM, CDH1, CDH2, and CTNNB1 ) in EECs and 12Z cells after IL-33 or/and ST2 neutralizing antibody treatment. D Protein expression of EMT-related genes (vimentin, E-cadherin, N-cadherin, and β-catenin) in EEC and 12Z treated by IL-33 or/and ST2 neutralizing antibody. E Protein expression of EMT-related genes (vimentin, E-cadherin, N-cadherin, and β-catenin) in EECs and 12Z cells treated by IL-33 or/and knocking down ST2 by siRNA. Data are presented as mean ± SEM. All data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test and Student’s t-test ; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The following IHC antibodies were used: anti-human ST2 (1:200), anti-human CCN4 (1:200), and anti-human E-cadherin (1:500) (abcam, Cambridge, UK); anti-human vimentin (1:100) (Cell Signaling Technology, Boston, USA); anti-human/mouse Fra-1 (1:200), anti-mouse CCN4 (1:100), and anti-mouse vimentin (1:100) (Affinity Biosciences, China); The primary antibodies used are listed in Supplementary Table 1.

Techniques: RNA Expression, Western Blot, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: The IL-33-ST2 axis plays a vital role in endometriosis via promoting epithelial–mesenchymal transition by phosphorylating β-catenin

doi: 10.1186/s12964-024-01683-x

Figure Lengend Snippet: IL-33 Enhances the Expression of Downstream Target Genes of WNT/β-catenin Pathway. A RNA expression of downstream target genes of WNT/β-catenin ( AXIN2, CCN4, CCND1, CD44, Fra-1, JUN, LGR5, MYC, and PPARD ) were detected via real-time PCR in EECs and 12Z cells. B Fra-1 expression in EECs and 12Z cells was detected by western blotting. C CCN4 secretion in the cell supernatant of EECs and 12Z cells was detected via ELISA. D - F After IL-33 or/and ST2 neutralizing antibody treatment, the mRNA expression and protein levels of Fra-1 and CCN4 in EECs and 12Z cells. G Immunohistochemical staining of Fra-1 and CCN4 in eutopic endometrium and ectopic lesions. Data are presented as mean ± SEM. All data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test and Student’s t-test ; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The following IHC antibodies were used: anti-human ST2 (1:200), anti-human CCN4 (1:200), and anti-human E-cadherin (1:500) (abcam, Cambridge, UK); anti-human vimentin (1:100) (Cell Signaling Technology, Boston, USA); anti-human/mouse Fra-1 (1:200), anti-mouse CCN4 (1:100), and anti-mouse vimentin (1:100) (Affinity Biosciences, China); The primary antibodies used are listed in Supplementary Table 1.

Techniques: Expressing, RNA Expression, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: The IL-33-ST2 axis plays a vital role in endometriosis via promoting epithelial–mesenchymal transition by phosphorylating β-catenin

doi: 10.1186/s12964-024-01683-x

Figure Lengend Snippet: IL-33 obviously Induced β-catenin Phosphorylation. A Protein expression of phospho-β-catenin (Ser675 and Ser552), phospho-CREB (Ser133), CREB, phospho-AKT(Ser473) and AKT in EECs and 12Z cells treated by IL-33 or/and ST2 neutralizing antibody. B Protein expression of phospho-β-catenin (Ser675 and Ser552), phospho-CREB (Ser133), CREB, phospho-AKT (Ser473), AKT, and Fra-1 in EECs and 12Z cells treated by IL-33 or/and knocking down ST2 by siRNA. C Expression of CCN4 in supernatant of EEC and 12Z treated by IL-33 or/and knocking down ST2 by siRNA. Data are presented as mean ± SEM. All data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test and Student’s t-test ; * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The following IHC antibodies were used: anti-human ST2 (1:200), anti-human CCN4 (1:200), and anti-human E-cadherin (1:500) (abcam, Cambridge, UK); anti-human vimentin (1:100) (Cell Signaling Technology, Boston, USA); anti-human/mouse Fra-1 (1:200), anti-mouse CCN4 (1:100), and anti-mouse vimentin (1:100) (Affinity Biosciences, China); The primary antibodies used are listed in Supplementary Table 1.

Techniques: Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: The IL-33-ST2 axis plays a vital role in endometriosis via promoting epithelial–mesenchymal transition by phosphorylating β-catenin

doi: 10.1186/s12964-024-01683-x

Figure Lengend Snippet: IL-33-ST2 Signal Blocking or β-catenin Knockout Weakens EMT in EEC and 12Z. A . Immunofluorescence staining of phospho-β-catenin at Ser675 and Ser552 (Scale bars, 20 μm). B , C Protein expression of EMT-related proteins (Vimentin, E-cadherin, N-cadherin, and β-catenin) and Fra-1 in EECs and 12Z cells treated by IL-33 or/and knocking β-catenin by siRNA. D Expression of CCN4 in supernatant of EEC and 12Z treated by IL-33 or/and knocking β-catenin by siRNA. Data are presented as mean ± SEM. All data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test and Student’s t-test ; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The following IHC antibodies were used: anti-human ST2 (1:200), anti-human CCN4 (1:200), and anti-human E-cadherin (1:500) (abcam, Cambridge, UK); anti-human vimentin (1:100) (Cell Signaling Technology, Boston, USA); anti-human/mouse Fra-1 (1:200), anti-mouse CCN4 (1:100), and anti-mouse vimentin (1:100) (Affinity Biosciences, China); The primary antibodies used are listed in Supplementary Table 1.

Techniques: Blocking Assay, Knock-Out, Immunofluorescence, Staining, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: The IL-33-ST2 axis plays a vital role in endometriosis via promoting epithelial–mesenchymal transition by phosphorylating β-catenin

doi: 10.1186/s12964-024-01683-x

Figure Lengend Snippet: IL-33 Promotes Fibrosis via EMT in Allograft Mouse Model of Endometriosis. A Schematic representation of the experimental outline shows the induction of endometriosis (day 0), i.p. injections of saline, IL-33, antiST2 or IL33 + anti ST2 every two days (beginning on day 3), and euthanasia (day 28). B Macroscopic view of ectopic endometriotic lesions in each group of mouse model. C , D Weight and number of lesions in mice treated with IL-33 or/and antiST2. E Body weights of mice in different groups. F - I . Masson, Fra1, vimentin, and CCN4 staining of lesions in different groups of mice. Data are presented as mean ± SEM. All data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test and Student’s t-test ; * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The following IHC antibodies were used: anti-human ST2 (1:200), anti-human CCN4 (1:200), and anti-human E-cadherin (1:500) (abcam, Cambridge, UK); anti-human vimentin (1:100) (Cell Signaling Technology, Boston, USA); anti-human/mouse Fra-1 (1:200), anti-mouse CCN4 (1:100), and anti-mouse vimentin (1:100) (Affinity Biosciences, China); The primary antibodies used are listed in Supplementary Table 1.

Techniques: Saline, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: The IL-33-ST2 axis plays a vital role in endometriosis via promoting epithelial–mesenchymal transition by phosphorylating β-catenin

doi: 10.1186/s12964-024-01683-x

Figure Lengend Snippet: Schematic illustration of IL-33 Promoting EMT Process in Endometriotic Milieu. Interleukin 33 (IL-33) is highly expressed in ectopic ESCs, acting via the receptor ST2. Ectopic milieu, characterized by ROS, TGF-β1, and high level of estrogen, triggers secretion of IL-33 in ESCs, which in turn, enhanced the aggressive implantation and survival of ESCs. Meanwhile, elevated IL-33 in ectopic milieu also activated WNT/β-catenin pathway in EECs by phosphorylating β-catenin (Ser675 and Ser552), which primes exresssion of ECM related genes (CCN4 and Fra-1) and mesenchymal markers, enhanceing the EMT process and extracellular matrix produnction. Thus, IL-33/ST2 axis plays a pivotal role in endometriosis progress by promoting EMT. This figure was created with biorender.com

Article Snippet: The following IHC antibodies were used: anti-human ST2 (1:200), anti-human CCN4 (1:200), and anti-human E-cadherin (1:500) (abcam, Cambridge, UK); anti-human vimentin (1:100) (Cell Signaling Technology, Boston, USA); anti-human/mouse Fra-1 (1:200), anti-mouse CCN4 (1:100), and anti-mouse vimentin (1:100) (Affinity Biosciences, China); The primary antibodies used are listed in Supplementary Table 1.

Techniques:

Journal: Cancer research

Article Title: IL33 Promotes Colon Cancer Cell Stemness via JNK Activation and Macrophage Recruitment

doi: 10.1158/0008-5472.CAN-16-1602

Figure Lengend Snippet: IL33 promotes colon cancer stemness via its receptor ST2. A, Expression of ST2 protein in colon cancer cells. Primary colon cancer cells (#1 and #2) and HT-29 cells were stained with specific rabbit anti-human ST2 Ab and R-PE–conjugated goat anti-rabbit IgG. The expression of ST2 was determined by flow cytometer analyzer and expressed as the percentage of ST2+ cells in total colon cancer cells. One of four experiments is shown. B, Expression of ST2 mRNA in colon cancer cells. ST2 mRNA expression was determined by RT-PCR in human umbilical vascular endothelial cells (HUVEC), peripheral blood mononuclear cells (PBMC), primary colon cancer cells, and HT-29 cells. Primary colon cancer cells and HT-29 cells were cultured with 100 ng/mL IL33 for 24 hours. One of three experiments is shown. C, Effects of anti-ST2 on the role of IL33-mediated colon cancer sphere formation. Primary colorectal cancer cells were subject to sphere assay. IL33 (100 ng/mL) and/or anti-ST2 antibody (1 μg/mL) were added in the sphere culture. Results are expressed as the mean ± SEM; n = 4; **, P < 0.01. D–F, Effects of anti-ST2 on the role of IL33-stimulated colon cancer stem cell gene expression. Primary colorectal cancer cells were cultured with IL33 (100 ng/mL) and/or anti-ST2 antibody (1 μg/mL) for 24 hours. The expression of NANOG (D), NOTCH3 (E), and OCT3/4 (F) transcripts were quantified by real-time PCR. Results are expressed as the mean ± SEM; n = 4; *, P < 0.05; **, P < 0.01.

Article Snippet: The NFκB inhibitor, BAY11-7082; the P38 inhibitor, SB203580; the JNK inhibitor, SP600125; the MEK/ERK inhibitor, PD98059 (Cayman Chemicals), and the iNOS inhibitor, SMT (Aladdin), and anti-human ST2 antibody (R&D Systems) were added in sphere culture for 1 week.

Techniques: Expressing, Staining, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Cancer research

Article Title: IL33 Promotes Colon Cancer Cell Stemness via JNK Activation and Macrophage Recruitment

doi: 10.1158/0008-5472.CAN-16-1602

Figure Lengend Snippet: IL33 promotes colon cancer stemness via recruiting and stimulating macrophages. A, Tumor-associated macrophages in IL33 transgenic mice. MC38 cells (106) were subcutaneously injected into wild type and IL33 transgenic mice. Tumor-infiltrating immune cells were stained for CD45 and F4/80 and were analyzed by FACS. Results are shown as the mean of F4/80+ macrophages ± SEM in CD45+ cells in day 28 tumor tissues; n = 4; *, P < 0.05. B, Effects of macrophage depletion on MC38 tumor growth. MC38 cells (106) were subcutaneously injected into wild type or IL33 transgenic mice. Clodronate liposomes were intraperitoneally injected into the mice. Tumor growth was monitored. Results are expressed as the mean of tumor volume ± SEM. – Møs, macrophage depletion; n = 4 per group; *, P < 0.05. C, Macrophage migration toward IL33. CD14+CD45+ macrophages were enriched and sorted from colon cancer tissues or normal blood and subjected to the migration assay in the presence of IL33 and/or anti-ST2. Results are expressed as the mean percentage of migrated cells ± SEM. Møs, macrophages. TAM, tumor associated macrophages; n = 4; *, P < 0.05. D, Effects of IL33-treated macrophages on colon cancer sphere formation. Normal blood CD14+ macrophages were treated with IL33 in the presence or absence of celecoxib for 72 hours. Primary colon cancer cells were subject to sphere formation in the presence of these treated macrophages. Results are expressed as the mean of sphere numbers ± SEM; n = 4 per group; *, P < 0.05. E, Effects of IL33 on macrophage-derived PGE2.Normal blood CD14+ macrophages were treated with IL33 for 48 hours. PGE2 was detected in the culture supernatants by ELISA. Results are expressed as the mean values ± SEM; n = 4 per group; *, P < 0.05. F, Effects of PGE2 on colon cancer sphere formation. Primary colon cancer cells were subject to sphere assay in the presence of PGE2 (50 ng/mL). Results are expressed as the mean of sphere numbers ± SEM; n = 4 per group; *, P < 0.01.

Article Snippet: The NFκB inhibitor, BAY11-7082; the P38 inhibitor, SB203580; the JNK inhibitor, SP600125; the MEK/ERK inhibitor, PD98059 (Cayman Chemicals), and the iNOS inhibitor, SMT (Aladdin), and anti-human ST2 antibody (R&D Systems) were added in sphere culture for 1 week.

Techniques: Transgenic Assay, Injection, Staining, Liposomes, Migration, Derivative Assay, Enzyme-linked Immunosorbent Assay