recombinant mouse il-33 ril-33  (PeproTech)

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 was upregulated and positively correlated with NET formation in renal transplant patients. ( A ) Paired analysis of serum IL-33 levels, ( B ) paired analysis of serum MPO-DNA levels, ( C ) paired analysis of serum citH3 levels in renal transplant patients preoperatively and postoperatively ( n = 20). ( D ) Correlation analysis for postoperative serum IL-33 level and serum MPO-DNA level (Spearman r = 0.6892, P = 0.0008). ( E ) Correlation analysis of postoperative serum IL-33 level and serum citH3 level (Spearman r = 0.6486, P = 0.002). * P < 0.05, ** P < 0.01

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques:

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 and NETs were elevated following renal I/R in mice. ( A - B ) Serum IL-33 levels ( A ) and renal tissue homogenate IL-33 levels ( B ) after renal I/R ( n = 6). ( C - D ) Representative blots ( C ) and statistical analysis ( D ) of IL-33 protein expression levels in renal tissues after I/R ( n = 6). ( E - F ) Representative images ( E ) and statistical analysis ( F ) of IL-33 immunohistochemistry in renal tissues after I/R ( n = 6). Scale bar was 50 μm. ( G - H ) IL-33 expression and distribution among the groups (blue staining indicate DAPI for nuclei), IL-33 (red) and CD31 (green) ( n = 6). Scale bar was 50 μm. (I-J) Serum MPO-DNA (I) and citH3 levels ( J ) following renal I/R ( n = 6). ( K - L ) Representative blots ( K ) and statistical analysis ( L ) of citH3 protein expression levels in renal tissues after I/R ( n = 6). ( M - N ) NET formation in in the indicated groups (blue indicate DAPI staining), MPO (red) and citH3 (green) ( n = 6). Scale bar was 20 μm. ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 promoted NET generation during renal IRI. ( A ) A diagram showing WT mice receiving renal IRI or sham surgery following intraperitoneal injection of rIL-33 (10 µg per mouse) or vehicle (PBS). ( B - C ) Serum MPO-DNA ( B ) and citH3 levels ( C ) of mice in each group ( n = 6). ( D - E ) Representative blots ( D ) and statistical analyses ( E ) of citH3 protein expression levels in renal tissues in the indicated groups ( n = 6). ( F - G ) NET formation in the indicated groups through DAPI staining (blue), MPO (red) and citH3 (green) ( n = 6). Scale bar was 20 μm. ( H - I ) Serum Cr ( H ) and BUN levels ( I ) in the indicated groups ( n = 6). ( J - K ) Representative blots ( J ) and statistical analysis ( K ) of KIM-1 protein expression levels in renal tissues in the indicated groups ( n = 6). ( L - N ) Renal tissues from mice in the indicated groups stained with HE and KIM-1 ( L ), followed by score of tubular injury ( M ) and quantitative analysis of KIM-1 ( N ) ( n = 6). Scale bar was 50 μm. * P < 0.05, ** P < 0.01

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Injection, Expressing, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 exacerbated renal IRI by inducing NET formation. ( A ) A diagram showing the time node of intraperitoneal injection of GSK484 (4 mg/kg) and rIL-33 (10 µg per mouse) in WT mice before renal I/R. (B-C) Serum MPO-DNA ( B ) and citH3 levels ( C ) in the indicated groups ( n = 6). ( D - E ) Representative blots ( D ) and statistical analysis ( E ) of citH3 expression levels in renal tissues of the indicated groups ( n = 6). ( F - G ) NET formation in the indicated groups as determined using the DAPI (blue), MPO (red) and citH3 (green) staining ( n = 6). Scale bar was 20 μm. ( H - I ) Serum Cr ( H ) and BUN levels ( I ) in the indicated groups ( n = 6). ( J - K ) Representative blots ( J ) and statistical analysis ( K ) of KIM-1 protein expression levels in renal tissues from the indicated groups ( n = 6). ( L - N ) Renal tissues of mice in the indicated groups stained with HE and KIM-1 ( L ), followed by score of tubular injury ( M ) and quantitative analysis of KIM-1 ( N ) ( n = 6). Scale bar was 50 μm. ** P < 0.01, *** P < 0.001

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Injection, Expressing, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: Blocking IL-33 improved renal IRI by reducing NET formation. ( A ) The diagram of WT mice receiving renal IRI or sham surgery after intraperitoneal injection of anti-IL-33 monoclonal antibody (Anti-IL-33) (10 µg per ounce) or vehicle (IgG2b Isotype Control). ( B - C ) Serum MPO-DNA ( B ) and citH3 levels ( C ) of mice in the indicated groups ( n = 6). (D-E) Representative blots ( D ) and statistical analysis ( E ) of citH3 protein expression levels in renal tissues in the indicated groups ( n = 6). ( F - G ) NET formation in the indicated groups following DAPI (blue), MPO (red) and citH3 (green) staining ( n = 6). Scale bar was 20 μm. ( H - I ) Serum Cr ( H ) and BUN levels ( I ) in the indicated group ( n = 6). ( J - K ) Representative blots ( J ) and statistical analysis ( K ) of KIM-1 protein expression levels in renal tissues in the indicated groups ( n = 6). ( L - N ) Renal tissues of mice in the indicated groups subjected to HE and KIM-1 staining ( L ), followed by score of tubular injury ( M ) and statistical analysis of KIM-1 ( N ) ( n = 6). Scale bar was 50 μm. ** P < 0.01, *** P < 0.001

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Blocking Assay, Injection, Control, Expressing, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 stimulated NET generation in vitro. ( A - B ) Neutrophils incubated with PBS, PMA (100nM) and various concentrations of rIL-33 (20, 60, 100 ng/mL) for 4 h. rIL-33 stimulation increased MPO-DNA ( A ) and citH3 levels ( B ) in the neutrophil culture medium in a dose-dependent manner relative to the PBS group ( n = 3). ( C - D ) Relative to the PBS group, rIL-33 activated neutrophils to increase citH3 protein in a dose-dependent manner ( n = 3). ( E ) Compared with the PBS group, rIL-33 increased the expression of ST2 mRNA on neutrophils ( n = 3). ( F ) Representative scanning electron microscopy graphs of neutrophils treated with PBS or rIL-33 (100ng/mL) for 4 h. Scale bar was 10 μm. ( G ) The NET formation as indicated by DAPI (blue), MPO (red) and citH3 (green) staining, was comparable between rIL-33 (100ng/mL) and PMA group. Scale bar was 50 μm. ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: In Vitro, Incubation, Expressing, Electron Microscopy, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 induced NET formation via ST2/PI3K/Akt and ST2/PAD4 signaling pathways. Mouse bone marrow-derived neutrophils were treated with PBS or rIL-33 (100ng/mL) for 4 h, followed by RNA sequencing ( n = 3). ( A - B ) Volcano plot ( A ) and heatmap ( B ) showing differential gene expression between PBS and rIL-33 groups. ( C ) GO enrichment analysis of DEGs. ( D ) KEGG pathway enrichment analysis of DEGs. Bone marrow-derived neutrophils from WT and ST2 KO mice were treated with PBS or rIL-33 (100ng/mL) for 4 h. The production of MPO-DNA ( E ) and citH3 ( F ) in the cell culture medium of ST2 KO neutrophils treated with IL-33 was markedly decreased relative to the IL-33-treated WT neutrophils ( n = 3). ( G ) Confocal microscopy was conducted to examine NET formation (co-localization of DAPI, MPO and citH3) in each group. ( H - I ) The expression of PI3K, p-PI3K, Akt, p-Akt and citH3 in WT and ST2 KO neutrophils stimulated by rIL-33 as determined by Western blot ( n = 3). ( J - K ) WT neutrophils were treated with 10µM LY294002 (PI3K inhibitor) or 10µM MK2206 (Akt inhibitor) for 24 h, and 100ng/mL rIL-33 was added on the 20th h to stimulate WT neutrophils for 4 h. The expression of PI3K, p-PI3K, Akt, p-Akt and citH3 in neutrophils was determined by Western blot ( n = 3). ( L ) Gene expression of Padi1, Padi2, Padi4 and Padi6 in neutrophils stimulated with rIL-33 ( n = 3). The gene expression data were expressed as log 2 (FPKM + 1). ( M - N ) PAD4 protein expression levels and quantitative analysis in WT and ST2 KO neutrophils treated with rIL-33 were quantified by Western blots ( n = 3). ( O - P ) WT neutrophils were pretreated with GSK484 (PAD4 inhibitor) for 30 min after treatment with 100ng/mL rIL-33 for 4 h. The protein expression of citH3 in neutrophils was determined by Western blot ( n = 3). ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Protein-Protein interactions, Derivative Assay, RNA Sequencing, Gene Expression, Cell Culture, Confocal Microscopy, Expressing, Western Blot

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: Schematic diagram of the mechanism by which IL-33 exacerbated renal IRI by enhancing NET formation during renal I/R

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques:

Journal: Immunology

Article Title: Interleukin‐33 alleviates psoriatic inflammation by suppressing the T helper type 17 immune response

doi: 10.1111/imm.13203

Figure Lengend Snippet: Interleukin‐33 (IL‐33) expression is markedly increased in individuals with moderate‐to‐severe psoriasis. (a) mRNA expression of IL‐33 in the skin of normal people (n = 16) and lesional skin of psoriasis patients (n = 16). (b) mRNA expression of IL‐17A in the skin of normal people (n = 6) and lesional skin of psoriasis patients (n = 6). (c) The correlation of the mRNA expression of IL‐33 and IL‐17A in the skin (n = 12). (d,e) Immunohistochemical staining of IL‐33 in the epidermis (d) and dermis (e) of skin of normal people (n = 10) and lesional skin of psoriasis patients (n = 10). (f) Serum level of IL‐33 of normal people (n = 15) and psoriasis patients (n = 25). (g) mRNA expression of IL‐33 and IL‐17A in the lesional of psoriasis patients before and after anti‐tumor necrosis factor‐α (TNF‐α) therapy (n = 5). (h) Serum level of IL‐33 of psoriasis patients before and after anti‐TNF‐α therapy (n = 10). Data show mean + SEM or mean ± SEM. P‐values were determined by unpaired Student’s t‐test. Correlation of the mRNA expression of IL‐33 and IL‐17A was determined by Pearson coefficient. **P < 0·01, ***P < 0·001 and ****P < 0·0001

Article Snippet: Mouse recombinant IL‐33 (rIL‐33, 1 µg in 300 μl PBS; PeproTech, Rocky Hill, NJ) or PBS was injected subcutaneously daily into the back skin of the mice.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Immunology

Article Title: Interleukin‐33 alleviates psoriatic inflammation by suppressing the T helper type 17 immune response

doi: 10.1111/imm.13203

Figure Lengend Snippet: Interleukin‐33 (IL‐33) suppresses T helper type 17 (Th17) cells in individuals with moderate‐to‐severe psoriasis. (a,b) The proportion of IL‐17A+ IFN‐γ –, IL‐17A+ IFN‐γ + and IL‐17A– IFN‐γ + cells and the geometric mean fluorescence intensity (gMFI) of IL‐17A and interferon‐γ (IFN‐γ) in CD4+ T cells from psoriasis patients (n = 17) treated with or without IL‐33 (50 ng/ml) for 72 hr. (c) The supernatant level of IL‐4 in CD4+ T cells from psoriasis patients (n = 10) treated with or without IL‐33 (50 ng/ml) for 72 hr. (d) The proportion of IL‐10+ and IFN‐γ + cells in Th17 (CD4+ and IL‐17A+) cells from psoriasis patients (n = 10) treated with or without IL‐33 (50 ng/ml) for 72 hr. (e) The proportion of regulatory T (Treg) (CD25+ and Foxp3+) cells in CD4+ T cells from psoriasis patients (n = 10) treated with or without IL‐33 (50 ng/ml) for 72 hr. (f) The proportion of invariant natural killer T (iNKT) (CD3+ and αGalCer‐CD1d+) cells in the peripheral blood mononuclear cells (PBMCs) from psoriasis patients (n = 10) treated with αGalCer and with or without IL‐33 (50 ng/ml) for 72 hr. Data show mean + SEM. P‐values were determined by paired Student’s t‐test. ns, no significance, *P < 0·05, **P < 0·01, ***P < 0.001 and ****P < 0.0001

Article Snippet: Mouse recombinant IL‐33 (rIL‐33, 1 µg in 300 μl PBS; PeproTech, Rocky Hill, NJ) or PBS was injected subcutaneously daily into the back skin of the mice.

Techniques: Fluorescence

Journal: Immunology

Article Title: Interleukin‐33 alleviates psoriatic inflammation by suppressing the T helper type 17 immune response

doi: 10.1111/imm.13203

Figure Lengend Snippet: Subcutaneous injection of interleukin‐33 (IL‐33) alleviates imiquimod (IMQ) ‐induced murine psoriatic inflammation. (a) Schematic representation of the plan with the injection of PBS or IL‐33 on mice treated with IMQ. (b) Representative photos of the lesional skin of mice from each group. (c) The disease severity scoring of the mice from each group based on scaling, erythema, and skin thickness from day 1 to day 7. (d) Representative hematoxylin & eosin staining of skin sections of mice from each group on day 7 (bar = 200 um). (e) Epidermal thickness and infiltrating inflammatory cells of the skin sections of mice from each group on day 7. Each group contains six mice and all experiments were repeated at least twice. Data show mean + SEM. P‐values were determined by one‐way analysis of variane with Bonferroni multiple‐comparisons t‐test. *P < 0·05, **P < 0·01 and ***P < 0·001

Article Snippet: Mouse recombinant IL‐33 (rIL‐33, 1 µg in 300 μl PBS; PeproTech, Rocky Hill, NJ) or PBS was injected subcutaneously daily into the back skin of the mice.

Techniques: Injection, Staining

Journal: Immunology

Article Title: Interleukin‐33 alleviates psoriatic inflammation by suppressing the T helper type 17 immune response

doi: 10.1111/imm.13203

Figure Lengend Snippet: Interleukin‐33 (IL‐33) altered the expression of inflammatory cytokines and T‐cell subpopulations of skin‐draining lymph nodes in imiquimod (IMQ) ‐treated mice. (a) The protein level of tumor necrosis factor‐α (TNF‐α), IL‐23, IL‐10 and interferon‐γ (IFN‐γ) of the skin of mice from each group. (b) The serum level of TNF‐α and IFN‐γ of the mice from each group. (c,d) The proportion of T‐cell subpopulations in the skin‐draining lymph nodes of mice from each group. (e) Immunohistochemical staining of CD3 in the skin of mice from each group. (f) Immunohistochemical staining of Ki67 in the skin of mice from each group. Each group contains six mice and all experiments were repeated at least twice. Data show mean + SEM. P‐values were determined by one‐way analysis of variance with Bonferroni multiple‐comparisons t‐test. *P < 0·05, **P < 0·01, ***P < 0·001 and ****P < 0·0001

Article Snippet: Mouse recombinant IL‐33 (rIL‐33, 1 µg in 300 μl PBS; PeproTech, Rocky Hill, NJ) or PBS was injected subcutaneously daily into the back skin of the mice.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Immunology

Article Title: Interleukin‐33 alleviates psoriatic inflammation by suppressing the T helper type 17 immune response

doi: 10.1111/imm.13203

Figure Lengend Snippet: Summary of interleukin‐33 (IL‐33) actions in psoriatic inflammation. IL‐17‐producing T cells [T helper type 17 (Th17), IL‐17‐producing γδT (γδT17) and IL‐17‐producing CD8+ T (Tc17) cells] produce and secrete large amounts of IL‐17A under the inflammatory microenvironment of psoriasis. Then IL‐17A acts on keratinocytes to promote the IL‐33 expression. On the one hand, the increased IL‐33 from keratinocytes or other cells in the dermis, such as fibroblasts, can promote the proliferation of keratinocytes to aggravate the psoriatic inflammation, but on the other hand, inhibit the differentiation and function of Th17 cells to remit the psoriatic inflammation

Article Snippet: Mouse recombinant IL‐33 (rIL‐33, 1 µg in 300 μl PBS; PeproTech, Rocky Hill, NJ) or PBS was injected subcutaneously daily into the back skin of the mice.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Interleukin-33 enhances programmed oncosis of ST2L-positive low-metastatic cells in the tumour microenvironment of lung cancer

doi: 10.1038/cddis.2015.418

Figure Lengend Snippet: Expression of ST2 in human normal lung tissues and lung cancers. ( A ) ST2 expression in lung adenocarcinomas (C) and in the adjacent normal lung tissues (N) based on the Hou and Bhattacharjee lung data sets in Oncomine database (Compendia Bioscience, Ann Arbor, MI, USA). ( B ) Correlation between ST2 expression and relapse-free survival, and overall survival in lung cancer patients. The data are based on the Okayama lung data set in PrognoScan database ( http://www.prognoscan.org ). ( C ) IL-33 expression in lung adenocarcinomas of different cancer stages based on the Okayama lung data set in Oncomine database. ( D ) Correlation between IL-33 expression and overall survival in lung cancer patients. The data are based on the Okayama lung data set in PrognoScan database. ( E ) Immunofluorescence staining of surfactant protein C (SP-C) and IL-33 in HLAEpiC cells. The nuclei were counterstained with DAPI. ( a ) Negative control (NC) for ( b ). Second antibody only; ( b ) SP-C. ( c ) NC for ( d ). ( d ) IL-33. Scale bars, 50 μ m. ( F ) qRT-PCR analysis of the expression of ST2L-related molecules in HLAEpiC cells and in human adenocarcinoma A549 cells. * P <0.05; ** P <0.002; *** P <0.0001. ( G ) RT-PCR analysis of the expression of IL-33/ST2L-related genes in human lung cancer cell lines. Adenocarcinomas: A549, PC-9 and PC-14 cells; squamous carcinoma: QG56, PC-1 and PC-10 cells; small-cell lung carcinoma: QG90 and PC-6 cells; bronchoalveolar cancer: H358 cells

Article Snippet: Recombinant mouse IL-33 (rIL-33) and recombinant mouse ST2L/IL-1 R4 Fc chimaera (rsST2L-Fc) were purchased from R&D Systems, Inc. (McKinley Place NE, MN, USA).

Techniques: Expressing, Immunofluorescence, Staining, Negative Control, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: Interleukin-33 enhances programmed oncosis of ST2L-positive low-metastatic cells in the tumour microenvironment of lung cancer

doi: 10.1038/cddis.2015.418

Figure Lengend Snippet: Expression of functional ST2L in the low-metastatic, but not in the high-metastatic, 3LL cells. ( a ) Western blot analysis of ST2L, IL-1RAcP and MyD88 protein expression. β -Actin served as the loading control. ( b ) Immunofluorescent staining of ST2L, IL-1RAcP and MyD88. Scale bars, 50 μm. The nuclei were counterstained with DAPI. ( c ) Activation of IL-33/ST2L signalling molecules in IL-33-treated P29 cells. P29 cells were treated with 100 ng/ml rIL-33 for the indicated times. β -Actin served as the loading control. *Note that rapid and transient IkB- α reduction was repeatedly observed after the rIL-33 treatment; the reason for this is unknown. See also and . ( d ) RT-PCR analysis of the mRNA expression of NF- κ B target genes in IL-33-treated P29 cells. P29 cells were treated with 100 ng/ml rIL-33 for the indicated times. Thioglycollate-elicited mouse peritoneal macrophages (Mφ) were used as the control. ( e ) In vitro growth of P29 cells cultured in the presence or absence of rIL-33 (100 ng/ml) in the regular medium. ( f ) Invasive ability of P29 cells treated with rIL-33 (100 ng/ml) for 2 days. The number of invaded cells per field is shown ( n =6). Bars, S.D. Western blot images ( a and c ) have been cropped for presentation. Uncropped images are provided in

Article Snippet: Recombinant mouse IL-33 (rIL-33) and recombinant mouse ST2L/IL-1 R4 Fc chimaera (rsST2L-Fc) were purchased from R&D Systems, Inc. (McKinley Place NE, MN, USA).

Techniques: Expressing, Functional Assay, Western Blot, Staining, Activation Assay, Reverse Transcription Polymerase Chain Reaction, In Vitro, Cell Culture

Journal: Cell Death & Disease

Article Title: Interleukin-33 enhances programmed oncosis of ST2L-positive low-metastatic cells in the tumour microenvironment of lung cancer

doi: 10.1038/cddis.2015.418

Figure Lengend Snippet: IL-33 content and IL-33-positive cells in P29 subcutaneous tumours. ( a ) IL-33 content in the lysates of normal tissues (liver, lung and spleen; n =3), A11 tumour tissues ( n =5) and P29 tumour tissues ( n =5). Bars, S.D. ( b ) IL-33 content in P29 tumour tissues established in B6-wild-type and in IL-33 −/− mice. ( c ) IL-33-positive cells in P29 tumours. Cryosections of P29 and A11 tumours were stained with the goat anti-IL-33 antibody followed by Alexa Fluor 488-conjugated chicken anti-goat IgG. The nuclei were counterstained with DAPI. The arrows show the cells with cytoplasmic and nuclear IL-33 staining. Scale bars, 50 μm. ( d ) The number of IL-33-positive cells per field (1 mm 2 ) in P29 tumour tissues established in B6-wild-type and in IL-33 −/− mice. n =20. ( e ) The effect of various cytokines on IL-33 expression in P29 and A11 cells. The cells were treated with vehicle alone, rIL-33 (10 ng/ml), rIL-1 β (10 ng/ml), rIL-4 (10 ng/ml), rIL-6 (10 ng/ml), rIFN- γ (10 ng/ml), rTNF- α (10 ng/ml) and rTRAIL (10 ng/ml) for 2 days. Total RNA was isolated and subjected to qRT-PCR. Bars: SD; * P <0.002; ** P <0.0002; *** P <0.0001. ( f ) Effect of rIL-1 β on IL-33 mRNA expression in P29 cells. P29 cells were treated with rIL-1 β at various concentrations for 2 days (left) or at 10 ng/ml for up to 3 days (right). Total RNA was isolated and subjected to qRT-PCR analysis. ( g ) Expression of the cytokines in P29 tumours. The peripheral (P), middle (M) and central (C) regions of P29 tumours established in B6-wild-type and in IL-33 −/− mice were resected, and the total RNA isolated from each region was subjected to qRT-PCR. Bars, S.D.

Article Snippet: Recombinant mouse IL-33 (rIL-33) and recombinant mouse ST2L/IL-1 R4 Fc chimaera (rsST2L-Fc) were purchased from R&D Systems, Inc. (McKinley Place NE, MN, USA).

Techniques: Staining, Expressing, Isolation, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Interleukin-33 enhances programmed oncosis of ST2L-positive low-metastatic cells in the tumour microenvironment of lung cancer

doi: 10.1038/cddis.2015.418

Figure Lengend Snippet: Enhancement of the cell death of the low-metastatic cells, but not the high-metastatic cells, after treatment with rIL-33 in nutrients-depleted medium and under hypoxic conditions. Cell viability was evaluated using the trypan blue exclusion (TBE) test unless otherwise indicated. ( a ) The low-metastatic (P29 and P34) and the high-metastatic (D6 and A11) cells were treated with rIL-33 (100 ng/ml) for 42 h in glucose-depleted (0.1 g/l; Gluc L ) medium. ( b ) P29 and A11 cells were treated with various concentrations of rIL-33 for 42 h in Gluc L medium. ( c ) P29 and A11 cells were treated with rIL-33 (100 ng/ml) for various periods in Gluc L medium. ( d ) P29 cells were treated with rIL-33 (100 ng/ml) for 42 h in medium containing various concentrations of glucose (0–0.4 g/l). ( e ) P29 cells were treated with rIL-33 (100 ng/ml) for 42 h in Gluc L medium. Cell viability was assessed by TBE test, MTT assay and clonogenic assay (Clono). ( f ) P29, P34, D6 and A11 cells were treated with rIL-33 (100 ng/ml) for 28 h in Gln − medium. ( g ) P29 cells were treated with rIL-33 (100 ng/ml) for 28 h in Gln − medium. Cell viability was assessed as described in ( e ). ( h ) P29 and A11 cells were treated with rIL-33 (100 ng/ml) for 42 h under hypoxic (1% O 2 ) or anoxic (<0.1% O 2 ) conditions. ( i ) P29 cells were treated with rIL-33 (100 ng/ml) for 42 h under FBS-depleted (0–0.5% FBS L ) conditions. Bars, S.D.; * P <0.05; ** P <0.003; *** P <0.001

Article Snippet: Recombinant mouse IL-33 (rIL-33) and recombinant mouse ST2L/IL-1 R4 Fc chimaera (rsST2L-Fc) were purchased from R&D Systems, Inc. (McKinley Place NE, MN, USA).

Techniques: MTT Assay, Clonogenic Assay

Journal: Cell Death & Disease

Article Title: Interleukin-33 enhances programmed oncosis of ST2L-positive low-metastatic cells in the tumour microenvironment of lung cancer

doi: 10.1038/cddis.2015.418

Figure Lengend Snippet: Involvement of ST2L in the IL-33-enhanced cell death of P29 cells. The cells were cultured for 38 h in the presence or absence of rIL-33 (100 ng/ml) in glucose-depleted (0.1 g/l; Gluc L ) medium. Cell viability was evaluated using the trypan blue exclusion test. ( a ) RT-PCR analysis of ST2L mRNA expression in P29 cells stably expressing control shRNA (shCont) and ST2L shRNA (shST2L #1 and #2). β -Actin (ACTB) served as the control. ( b ) Sensitivity of shCont, shST2L #1 and shST2L #2 cells to rIL-33 (100 ng/ml). ( c ) Western blot analysis of the expression of MyD88 protein in P29 cells transiently transfected with control siRNA (siCont) or with MyD88 siRNA (siMyD88). Western blot images have been cropped for presentation. Uncropped images are provided in . ( d ) Sensitivity of siCont and siMyD88 cells to rIL-33 (100 ng/ml). ( e ) Secretion of sST2 by P29 cells stably expressing control shRNA or sST2 shRNA. The indicated cells were cultured for 24 h and the amount of sST2 in the conditioned medium was quantified by ELISA. ( f ) Sensitivity of shCont and shsST2 cells to rIL-33 (100 ng/ml). ( g ) Effect of an anti-ST2 antibody on the sensitivity of P29 cells to rIL-33 (100 ng/ml). P29 cells were cultured in the presence of control goat IgG or anti-ST2 antibody (1 μg/ml). Bars, S.D. * P <0.04; ** P <0.01; *** P <0.001. NS, not significant

Article Snippet: Recombinant mouse IL-33 (rIL-33) and recombinant mouse ST2L/IL-1 R4 Fc chimaera (rsST2L-Fc) were purchased from R&D Systems, Inc. (McKinley Place NE, MN, USA).

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Stable Transfection, shRNA, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Interleukin-33 enhances programmed oncosis of ST2L-positive low-metastatic cells in the tumour microenvironment of lung cancer

doi: 10.1038/cddis.2015.418

Figure Lengend Snippet: Analysis of IL-33/ST2L signalling pathways involved in IL-33-enhanced P29 cell death. ( a – c ) Western blot analysis of the effect of IL-33 on the phosphorylation of signalling molecules in P29 cells. P29 cells were cultured with rIL-33 (100 ng/ml) in glucose-depleted (0.1 g/l; Gluc L ) medium for the indicated times ( a and b ) or for 1 h ( c ). β -Actin served as the loading control. ( d ) Effect of various inhibitors on rIL-33-enhanced cell death. P29 cells were cultured with rIL-33 (100 ng/ml) in Gluc L medium for 42 h with SB203580 (20 μ M), SP600125 (20 μ M), wortmannin (10 μ M), BAY11-7082 (5 μ M) or rapamycin (1 μ M). Vehicle (DMSO) was added to the control culture. Cell viability was evaluated using the trypan blue exclusion test. Bars; S.D.; * P <0.005: ** P <0.002. NS, not significant. Western blot images ( a–c ) have been cropped for presentation. Uncropped images are provided in

Article Snippet: Recombinant mouse IL-33 (rIL-33) and recombinant mouse ST2L/IL-1 R4 Fc chimaera (rsST2L-Fc) were purchased from R&D Systems, Inc. (McKinley Place NE, MN, USA).

Techniques: Western Blot, Cell Culture

Journal: Cell Death & Disease

Article Title: Interleukin-33 enhances programmed oncosis of ST2L-positive low-metastatic cells in the tumour microenvironment of lung cancer

doi: 10.1038/cddis.2015.418

Figure Lengend Snippet: Induction of oncosis in IL-33-treated P29 cells under glucose-depleted conditions. ( a ) Effect of zVAD-fmk on rIL-33-induced cell death. P29 cells were treated with or without zVAD-fmk (10 μ M) in the presence or absence of rIL-33 (100 ng/ml) under Gluc L or Gln − conditions for 42 or 28 h, respectively. Bars, S.D.; * P <0.002. ( b ) Effect of necrostatin-1 on rIL-33-induced cell death. P29 cells were treated with or without necrostatin-1 (Nec-1; 10 μ M) in the presence or absence of rIL-33 (100 ng/ml) in Gluc L medium or in Gln − medium for 42 or 28 h, respectively. Bars, S.D. * P <0.002. ( c ) Conversion of LC3-I to LC3-II. P29 and A11 cells were incubated in the presence or absence of rIL-33 (100 ng/ml) under Gluc L or Gln − conditions for 42 or 28 h, respectively. ( d ) Appearance of cells with cytoplasmic blisters. P29 and A11 cells were treated with or without rIL-33 (100 ng/ml) for 40 h in glucose-depleted (0.1 g/l; Gluc L ) medium. Arrows and arrowheads indicate the cells with blisters and those cells with blebs, respectively. Scale bars, 50 μ m. ( e ) Percentage of cells with cytoplasmic blisters. P29 cells were treated with or without rIL-33 (100 ng/ml) for 36 h in Gluc L medium. Bars, S.D. * P <0.002. ( f ) Appearance of the cells with karyolysis. P29 and A11 cells were treated with or without rIL-33 (100 ng/ml) for 42 h in Gluc L medium. The nuclei were stained with DAPI. Arrows and arrowheads indicate cells with karyolysis and with apoptotic nuclei, respectively. Scale bars, 100 μ m (white); 5 μ m (yellow). ( g ) Percentage of cells with karyolysis. P29 cells were treated with or without rIL-33 (100 ng/ml) for 36 or 42 h in Gluc L medium. Bars, S.D. * P <0.002. ( h ) Effect of rIL-33 on ROS production in P29 cells. P29 cells were cultured with or without rIL-33 (100 ng/ml) in Gluc L or Gln − medium for 30 or 20 h, respectively. ROS production was measured by flow cytometry after staining the cells with H 2 DCF-DA. ( i ) Effect of N -acetylcysteine (NAC) on rIL-33-induced cell death. P29 cells were cultured with rIL-33 (100 ng/ml) in the presence or absence of NAC (10 mM) for 42 h. * P <0.003. NS, not significant

Article Snippet: Recombinant mouse IL-33 (rIL-33) and recombinant mouse ST2L/IL-1 R4 Fc chimaera (rsST2L-Fc) were purchased from R&D Systems, Inc. (McKinley Place NE, MN, USA).

Techniques: Incubation, Staining, Cell Culture, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Interleukin-33 enhances programmed oncosis of ST2L-positive low-metastatic cells in the tumour microenvironment of lung cancer

doi: 10.1038/cddis.2015.418

Figure Lengend Snippet: IL-33 suppresses P29 tumour growth and stimulates A11 selection under conditions mimicking the tumour microenvironment. ( a ) Tumour growth of P29 and A11 cells in B6-wild-type and in IL-33 −/− mice. P29 and A11 cells (1 × 10 5 cells) were subcutaneously injected into B6-wild-type (B6) and IL-33 −/− (KO) mice. n =7. Bars, S.D. ( b ) Gross observation of P29 tumours established by P29 cells in B6-wild-type and IL-33 −/− mice. The tumours were divided into two equal portions, and the divided faces are shown. ( c ) RT-PCR analysis of the expression of ST2L and sST2 in P29 cells stably expressing control shRNA (shCont) and ST2 shRNA (shST2 #2). ( d ) shCont and shST2 #2 cells (4 × 10 5 cells) were subcutaneously injected in B6 mice. n =7. Bars, S.D. ( e ) Effect of rIL-33 on A11 cell selection. A 1 : 1 mixture of EGFP-P29 and DsRed-A11 cells was cultured with or without rIL-33 (100 ng/ml) for 3 days under Gluc L /H/FBS L conditions and then for an additional 2 days in regular medium (recovery). Bars, S.D. * P <0.01; ** P <0.001. NS, not significant. ( f ) rIL-33 stimulation of an A11 selection under conditions mimicking the tumour microenvironment. A 1 : 1 mixture of EGFP-P29 and DsRed-A11 cells was cultured as described in ( e ). The cells were observed under a confocal laser microscope. Bars, 100 μ m

Article Snippet: Recombinant mouse IL-33 (rIL-33) and recombinant mouse ST2L/IL-1 R4 Fc chimaera (rsST2L-Fc) were purchased from R&D Systems, Inc. (McKinley Place NE, MN, USA).

Techniques: Selection, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Stable Transfection, shRNA, Cell Culture, Microscopy