Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: IL-33 and ST2L are overexpressed in human GC tissues and predict poor prognosis. a QRT-PCR analysis of IL-33 mRNA expression in GC and corresponding normal tissues ( n = 18). b Histogram displaying the relative mRNA expression of IL-33 in 18 GC tissues. Data are shown as −ΔΔCt and 2 −ΔCt . c IHC staining of α-SMA, IL-33 and ST2L in GC tissues (200×; scale bar = 100 μm). d Histogram displaying the number of α-SMA, IL-33 and ST2L positive cells/field in GC tissues. e Histogram displaying the correlation between IL-33 expression and ST2L expression determined by IHC ( r = 0.6503). f , g Survival of patients with low or high levels of IL-33 and ST2L protein expression. h – j Immunofluorescence staining of IL-33, α-SMA, FAP, ST2L and Cytokeratin in GC tissues (200×; scale bar = 400 μm). k – m Histogram displaying the percentage of IL-33 + /α-SMA + , IL-33 + /FAP + and ST2L + /Cytokeratin ++ cells in GC tissues. Data are represented as the mean ± SD; * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Quantitative RT-PCR, Expressing, Immunohistochemistry, Immunohistochemistry-Resin, Immunofluorescence, Staining

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: Relationship between IL-33 and ST2L expression levels and clinicopathologic parameters in 134 GC tissues

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Expressing, Immunostaining

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: CAFs-derived IL-33 promotes the migration, invasion and EMT of GC cells. a – d The migration and invasion ability of SGC7901 and MKN45 cells were analyzed after culture in medium alone (blank) or treatment with: exogenous IL-33 (300 ng/ml); co-culture with CAFs supplemented with IgG isotype antibody (3 μg/ml) or IL-33 neutralizing antibody (3 μg/ml). Histograms show the average cell number/field (100×; scale bar = 100 μm). e – h The migration and invasion ability of SGC7901 and MKN45 cells was detected after culture in medium alone (blank), co-culture with CAFs transfected with IL-33/siRNA or nc/siRNA. Histograms display the average cell number/field (100×; scale bar = 100 μm). i – l The migration and invasion ability of SGC7901 and MKN45 cells was analyzed after culture in medium alone (blank) or stimulation with exogenous IL-33 (300 ng/ml) plus IgG isotype antibody or ST2L neutralizing antibody (3 μg/ml); co-culture with CAFs in the presence of IgG isotype antibody (3 μg/ml) or ST2L neutralizing antibody (3 μg/ml). Histograms show the average cell number/field. (100×; scale bar = 100 μm). m , n QRT-PCR of the genes for EMT in SGC7901 and MKN45 cells after culture in medium alone (blank); or activation with exogenous IL-33 (300 ng/ml); co-culture with CAFs in the presence of IgG isotype antibody (3 μg/ml) or IL-33 neutralizing antibody (3 μg/ml). o – r Western blot of EMT markers in SGC7901 and MKN45 cells cultured in medium alone (blank); or stimulated with exogenous IL-33 (300 ng/ml) or co-culture with CAFs or co-cultured with CAFs in the presence of DMSO, U0126 (20 μM), IL-33 neutralizing antibody (3 μg/ml) or ST2L neutralizing antibody (3 μg/ml). p and r Densitometric analysis shows the expression level of EMT markers in GC cells stimulated by the above factors. Data are represented as the mean ± SD of three independent experiments; * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Derivative Assay, Migration, Co-Culture Assay, Transfection, Quantitative RT-PCR, Activation Assay, Western Blot, Cell Culture, Expressing

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: The metastatic potential of GC cells induced by CAFs-derived IL-33 is mediated by the activation of ERK1/2-SP1-ZEB2 pathway via ST2L. a – d Western blot analysis showing the phosphorylation of ERK1/2 in SGC7901 and MKN45 cells treated with medium alone or stimulated as follows: exogenous IL-33 (300 ng/ml), U0126 (20 μM), CAFs supernatants (CAFsu) supplemented with IgG isotype control antibody (3 μg/ml), DMSO; co-cultured with CAFs transfected with IL-33/siRNA or NC/siRNA; CAFsu supplemented with IL-33 neutralizing antibody (3 μg/ml). e Western blot analysis of pERK1/2 in SGC7901 and MKN45 cells incubated with medium alone; exogenous IL-33 (300 ng/ml); U0126 (20 μM); co-culture with CAFs; IL-33 neutralizing antibody (3 μg/ml); and/or ST2L neutralizing antibody (3 μg/ml). f , g The migration and invasion ability of SGC7901 and MKN45 cells after culture in medium alone (blank) or stimulation with exogenous IL-33 (300 ng/ml) and/or U0126 (20 μM); co-culture with CAFs and/or U0126 (20 μM). Histograms show the average cell number per field. (100×; scale bar = 100 μm). h , i QRT-PCR analysis of SP1 mRNA expression in SGC7901 and MKN45 cells incubated in medium alone or stimulation with exogenous IL-33 (300 ng/ml) or CAFs in the presence of IgG isotype control antibody (3 μg/ml) or IL-33 neutralizing antibody (3 μg/ml). j , k Western blot analysis of pERK1/2, ERK1/2, SP1 and ZEB2 in SGC7901 and MKN45 cells incubated in medium alone or stimulated by co-culture with CAFs in the presence of exogenous IL-33 (300 ng/ml) or U0126 (20 μM). l Western blot analysis showing the protein expression of pERK1/2, ERK1/2, SP1 and ZEB2 in SGC7901 and MKN45 cells cultured in medium alone (Mock); or transfected with control plasmid (nc) or SP1-expressing plasmid. m Dual luciferase reporters containing four different lengths of the ZEB2 promoter were co-transfected with SP1-expressing plasmid into 293T cells. Firefly luciferase activity was assessed relative to Renilla luciferase activity. Data are representative of three independent experiments. Densitometry shows relative protein expression. * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Derivative Assay, Activation Assay, Western Blot, Phospho-proteomics, Control, Cell Culture, Transfection, Incubation, Co-Culture Assay, Migration, Quantitative RT-PCR, Expressing, Plasmid Preparation, Luciferase, Activity Assay

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: Knockdown of IL-33 expression in CAFs or ST2L expression in GC cells inhibits peritoneal dissemination of GC cells in nude mice. a , c In vivo tumor peritoneal dissemination was examined by co-injection of SGC7901 or MKN45 cells and CAFs transfected with control siRNA (NC) or IL-33 siRNA into nude mice. Metastatic nodules are indicated by the red arrows. b , d Histograms displaying the number of peritoneal nodules in nude mice. e – h In vivo tumor peritoneal dissemination was examined by co-injection of CAFs and GC cells with ST2L knockdown (KD) into nude mice. Metastatic nodules are indicated by the red arrows. f , h Histograms displaying the number of peritoneal nodules in nude mice. Data are representative of three independent experiments. * P <0.05, * P <0.05, ** P <0.01

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Knockdown, Expressing, In Vivo, Injection, Transfection, Control

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: Model for the epithelial–stromal interactions in the tumor microenvironment of GC. a IL-33 released from CAFs promotes the migration, invasion and EMT of GC cells via the ST2L-ERK1/2-SP1-ZEB2 pathway. b GC cell-derived TNF-α upregulates IL-33 expression in CAFs via the TNFR2-NF-κB-IRF-1 pathway

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Migration, Derivative Assay, Expressing

Journal: Cell reports

Article Title: Organoids capture tissue-specific innate lymphoid cell development in mice and humans.

doi: 10.1016/j.celrep.2022.111281

Figure Lengend Snippet: Figure 3. SIOs provide an essentially GF model of gut-specific ILCs (A) Representative flow plots of NKp46 expression in ILCP + SIO co-culture-derived ILCs or SI-LP-derived CD127+ ILCs, with the frequency of NKp46+ ILC3s (co- culture: Live, EpCAM, Lin, CD45+, RORgt+; primary tissue: Live, CD45+, Lin, CD127+, Klrg1, NK1.1+/, RORgt+) additionally quantified for ILCPs cultured without SIOs or with GF SIOs in (B) (N = 2–5 animals, pooled from two experiments). (C) Relative frequency of mature ILC subsets excluding immature or other cells, depicting group 1 (magenta; Live, EpCAM, CD45+, Lin, RORgt-, ST2, Klrg1, NK1.1+, NKp46+), group 2 (green; Live, EpCAM, CD45+, Lin, RORgt, NK1.1, ST2+, Klrg1+, Sca-1+), NKp46+ group 3 (lavender; Live, EpCAM, CD45+, Lin, ST2, Klrg1, RORgt+, NKp46+), and NKp46 group 3 ILCs (blue; Live, EpCAM, CD45+, Lin, ST2, Klrg1, RORgt+, NKp46) in live, unstimulated co-cultures derived from SPF-SIOs or GF-SIOs compared with primary SPF ileum (no Peyer’s patches). (D) Diagram of transwell culture strategy. (E) Relative frequency of group 1, 2, and 3 ILCs derived from PD-1+ ILCP + SIO +/ transwell insert (TW) separation (N = 3, two experiments).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human CRTh2 – PE Miltenyi Biotec Cat# 130-113-600 Human CRTh2– BV711 BioLegend Cat# 350124 Human CRTh2 – BV421 BioLegend Cat# 350112 Human CD161 APC, A700 BioLegend Cat# 302012 Human ST2 – APC R&D Sys Cat# FAB5231A Human ST2 – PE R&D Sys Cat# FAB5231P Human NKp44 – PE Cy7 BioLegend Cat# 325116 Human NKp44 – PerCP Cy5.5 BioLegend Cat# 325114 Human/Mouse Tbet – PE-Cy7 BioLegend Cat# 644824 Human RORgt – APC Thermofisher (eBioscience) Cat# 17-6988-82 Human RORgt – PE BDBiosciences Cat# 563081 Human GATA3 – APC Cy7 Santa Cruz Cat# sc-268 Human IL22 – PerCP Cy5.5 BioLegend Cat# 366709 Human IL17A – PE-Dazzle BioLegend Cat# 512335 Human IL17A – e450 BD Horizon/ bioscience Cat# 560610 Human IL17A – BV786 BD Horizon/ bioscience Cat# 563745 Human IFNg –APCe780 Invitrogen Cat# 47-7319-41 Human IL-5 – APC BioLegend Cat# 504305 Human IL-13 – FITC eBioscience Cat# 11-7139-41 Human IL-13 – bv711 BD Biosciences Cat# 564288 Human CD25 – PerCP-Cy 5.5 BD Biosciences Cat# 560503 Human Klrg1 – APC Thermofisher (eBioscience) Cat# 25-5893-80 Human CCR6 – APC BioLegend Cat# 353416 Human CCR6 – BV605 BioLegend Cat# 353419 Human NKp46 APC BioLegend Cat# 331918 FcR CD16/32 blocking, mouse BioCell Cat# BE0307 FcR blocking, human Miltenyi Biotec Cat# 130-059-901 Anti-rhIL33 (neutralising, ICC, goat polyclonal) R&D Cat# AF3625 Anti-rmIL33 (neutralising, ICC, goat polyclonal) R&D Cat# AF3626 E-Cadherin – anti-human (rat) Thermofisher (eBioscience) Cat# 51-3249-82 CDX2 – anti-human (rabbit) abcam Cat# Ab76541 EpCAM – anti-mouse (rabbit) abcam Cat# Ab71916 CD45 anti-human (mouse) BioLegend Cat# 304001 ZO-1 anti-mouse (rabbit) Abcam Cat# Ab96587 Dclk1 anti-mouse (rabbit) Abcam Cat# Ab31704 CD44 anti-mouse/human (rat) Thermofisher (eBioscience) Cat# 14-0551-82 Critical commercial assays CellTrace FarRed ThermoFischer Cat# C34564 Foxp3 / Transcription Factor Staining Buffer Set Invitrogen eBioscience Cat# 00-5523-00 Live Dead fixable blue/UV ThermoFischer Cat# L34961 UltraComp eBeads Invitrogen Cat# 01-2222-42 RNeasy Qiagen Cat# 74106 RevertAid First Strand cDNA Synthesis Kit ThermoFisher Cat# K1622 Fast SYBR green master mix Applied Biosystems Cat# 4385612 (Continued on next page) Cell Reports 40, 111281, August 30, 2022 e2

Techniques: Expressing, Co-Culture Assay, Derivative Assay, Cell Culture

Journal: Cell reports

Article Title: Organoids capture tissue-specific innate lymphoid cell development in mice and humans.

doi: 10.1016/j.celrep.2022.111281

Figure Lengend Snippet: Figure 7. Gut-matured ILC2 upregulates ST2 on transfer to HLO culture (A) Representative image of SD-HIOs and SD-HLOs showing E-cadherin+ (E-CAD) epithelium, CD45+ ILCs, and nuclei (Hoechst) after 14-day co-culture (scale bars: 50 mm). (B) Count of EpCAM, CD45+, LIN ILCs after 14-day co-culture with SD-HIOs or SD-HLOs, with corresponding count of EpCAM, CD45 mesenchyme.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human CRTh2 – PE Miltenyi Biotec Cat# 130-113-600 Human CRTh2– BV711 BioLegend Cat# 350124 Human CRTh2 – BV421 BioLegend Cat# 350112 Human CD161 APC, A700 BioLegend Cat# 302012 Human ST2 – APC R&D Sys Cat# FAB5231A Human ST2 – PE R&D Sys Cat# FAB5231P Human NKp44 – PE Cy7 BioLegend Cat# 325116 Human NKp44 – PerCP Cy5.5 BioLegend Cat# 325114 Human/Mouse Tbet – PE-Cy7 BioLegend Cat# 644824 Human RORgt – APC Thermofisher (eBioscience) Cat# 17-6988-82 Human RORgt – PE BDBiosciences Cat# 563081 Human GATA3 – APC Cy7 Santa Cruz Cat# sc-268 Human IL22 – PerCP Cy5.5 BioLegend Cat# 366709 Human IL17A – PE-Dazzle BioLegend Cat# 512335 Human IL17A – e450 BD Horizon/ bioscience Cat# 560610 Human IL17A – BV786 BD Horizon/ bioscience Cat# 563745 Human IFNg –APCe780 Invitrogen Cat# 47-7319-41 Human IL-5 – APC BioLegend Cat# 504305 Human IL-13 – FITC eBioscience Cat# 11-7139-41 Human IL-13 – bv711 BD Biosciences Cat# 564288 Human CD25 – PerCP-Cy 5.5 BD Biosciences Cat# 560503 Human Klrg1 – APC Thermofisher (eBioscience) Cat# 25-5893-80 Human CCR6 – APC BioLegend Cat# 353416 Human CCR6 – BV605 BioLegend Cat# 353419 Human NKp46 APC BioLegend Cat# 331918 FcR CD16/32 blocking, mouse BioCell Cat# BE0307 FcR blocking, human Miltenyi Biotec Cat# 130-059-901 Anti-rhIL33 (neutralising, ICC, goat polyclonal) R&D Cat# AF3625 Anti-rmIL33 (neutralising, ICC, goat polyclonal) R&D Cat# AF3626 E-Cadherin – anti-human (rat) Thermofisher (eBioscience) Cat# 51-3249-82 CDX2 – anti-human (rabbit) abcam Cat# Ab76541 EpCAM – anti-mouse (rabbit) abcam Cat# Ab71916 CD45 anti-human (mouse) BioLegend Cat# 304001 ZO-1 anti-mouse (rabbit) Abcam Cat# Ab96587 Dclk1 anti-mouse (rabbit) Abcam Cat# Ab31704 CD44 anti-mouse/human (rat) Thermofisher (eBioscience) Cat# 14-0551-82 Critical commercial assays CellTrace FarRed ThermoFischer Cat# C34564 Foxp3 / Transcription Factor Staining Buffer Set Invitrogen eBioscience Cat# 00-5523-00 Live Dead fixable blue/UV ThermoFischer Cat# L34961 UltraComp eBeads Invitrogen Cat# 01-2222-42 RNeasy Qiagen Cat# 74106 RevertAid First Strand cDNA Synthesis Kit ThermoFisher Cat# K1622 Fast SYBR green master mix Applied Biosystems Cat# 4385612 (Continued on next page) Cell Reports 40, 111281, August 30, 2022 e2

Techniques: Co-Culture Assay

Journal: JCI insight

Article Title: From proteomics to discovery of first-in-class ST2 inhibitors active in vivo.

doi: 10.1172/jci.insight.99208

Figure Lengend Snippet: Figure 1. Flowchart of integrated high-throughput screening and computational analysis for discovery of ST2 inhibitors. PAINS, pan-assay interference compounds; MACCS, Molecular ACCess System; hPBMC, human peripheral blood mononuclear cell.

Article Snippet: The well with 3 nM ST2 alone was used as a negative control, whereas the ST2/IL-33 mixture with a human ST2 antibody (R&D Systems, MAB523) added at 0.45 ng/μl was used as the positive control.

Techniques: High Throughput Screening Assay, Pan Assay

Journal: JCI insight

Article Title: From proteomics to discovery of first-in-class ST2 inhibitors active in vivo.

doi: 10.1172/jci.insight.99208

Figure Lengend Snippet: Figure 3. IC50 values of iST2-1 and its analogs, SAXS studies of apo-ST2, ST2/iST2-1, and potential iST2-1 binding sites identified from computational simulations. (A) Chemical structures of racemic iST2-1, (R)-iST2-1, (S)-iST2-1, analogs of iST2-1, and structural alignment of (R)-iST2-1 (purple) with (S)-iST2-1 (green). IC50 values of the inhibitors are shown in parentheses. (B) Inhibition curves and IC50 values (in parentheses) of 4 representative compounds. Each data point is an average of triplicate measurements ± SD. (C) SAXS profiles, the residual plot between the SAXS profiles of apo-ST2 and ST2/iST2-1, comparison of the Kratky plot based on the SAXS profiles of apo-ST2 and ST2/iST2-1, the pair-wise distance distribution [P(r)] of apo-ST2 and ST2/iST2-1 calculated from the SAXS profiles. Dmax values are shown in parentheses. (D) Ab initio shape reconstruction of apo-ST2 (gray) and ST2/iST2-1 (purple). (E) Mapping of the (R)- iST2-1 and (S)-iST2-1 binding sites (surface envelop) in ST2 based on 32-ns MD simulations. Maps detected from 13- and 15-Å octahedron boxes are colored in purple and green, respectively. Consensus binding sites (S1r, S2r and S1s) are circled. The D1 and D2 domains of ST2 are labeled. (F) Binding sites of (R)-iST2-1 (green mesh) and (S)-iST2-1 (red mesh) that block interaction between ST2 and IL-33 (orange) are highlighted. (G) Binding sites of (R)-, (S)-, (S,S)-iST2-1 in the glycosylated ST2 from the MD simulations. (S,S)-iST2-1 is (S)-iST2-1 in which the proton on the pyrrolidine group is in the S form and its mapped site is shown in cyan mesh. SAXS, small-angle X-ray scattering.

Article Snippet: The well with 3 nM ST2 alone was used as a negative control, whereas the ST2/IL-33 mixture with a human ST2 antibody (R&D Systems, MAB523) added at 0.45 ng/μl was used as the positive control.

Techniques: Binding Assay, Inhibition, Comparison, Labeling, Blocking Assay

Journal: JCI insight

Article Title: From proteomics to discovery of first-in-class ST2 inhibitors active in vivo.

doi: 10.1172/jci.insight.99208

Figure Lengend Snippet: Figure 4. Dosages and treatment schedule for ST2 inhibitors and ex vivo analysis in the GVHD mouse models. (A) The dosages were calcu- lated using the body weight of each mouse at 20 mg and correspond to twice the IC50 values of the inhibitors determined by the HEK-Blue IL-33 assay. (B) Number of T cells infiltrat- ing the gut in the B6C3H.SW GVHD model at day 14. Flow cytometric analysis of intestinal CD4+IFN-γ+ and FoxP3+CD4+ T cell populations on day 14 in the (C) hu-T cells → NSG model and day 21 in the (D) hu-T cells → NSG, (E) B6 → C3H.SW GVHD models treat- ed with iST2-1, iST2-2, or iST2-3. n = 2 per group at day 14 and 21 respectively, and for each model. Data represent mean ± SEM (n = 2). TBI, total body irradiation; hPBMC, human peripheral blood mononuclear cell. *P < 0.05, **P < 0.01 by unpaired t test.

Article Snippet: The well with 3 nM ST2 alone was used as a negative control, whereas the ST2/IL-33 mixture with a human ST2 antibody (R&D Systems, MAB523) added at 0.45 ng/μl was used as the positive control.

Techniques: Ex Vivo, Irradiation

Journal: JCI insight

Article Title: From proteomics to discovery of first-in-class ST2 inhibitors active in vivo.

doi: 10.1172/jci.insight.99208

Figure Lengend Snippet: Figure 5. sST2 and IFN-γ levels, GVHD scores, and survival curves for GVHD disease model mice treated with ST2 inhibitors. (A) Plasma levels of human and murine sST2 and IFN-γ in the hu-T cells → NSG and B6 → C3H. SW GVHD models from day 7 to 28. Data represent mean ± SEM (n = 3 per group for NSG and n = 6 for C3H. SW). (B) GVHD scores and survival curves for the hu-T cells → NSG and B6 → C3H.SW GVHD models. Data rep- resent mean ± SEM for GVHD scores, and Kaplan-Meier curves for survival (n = 13 per group). P values were calculated for GVHD scores by unpaired t test and for survival by log-rank test. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The well with 3 nM ST2 alone was used as a negative control, whereas the ST2/IL-33 mixture with a human ST2 antibody (R&D Systems, MAB523) added at 0.45 ng/μl was used as the positive control.

Techniques: Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Increased Dysfunctional and Plastic Regulatory T Cells in Idiopathic Orbital Inflammation

doi: 10.3389/fimmu.2021.634847

Figure Lengend Snippet: IL-33 restored the suppressive function of Tregs, inhibited the production of interferon (IFN)-γ by regulatory T cells (Tregs) and the activation of orbital fibroblasts (OFs) in idiopathic orbital inflammation (IOI). (A) The plasma level of interleukin (IL)-33 in healthy controls (n = 18) and patients with IOI (n = 22) was assessed by Luminex technology. (B) The proportion of circulating ST2 + Foxp3 + Tregs in healthy controls (n = 14) and patients with IOI (n = 8). (C) IL-33 and (D) IL1RL1 mRNA expression in orbital fat from healthy subjects (n = 4) and patients with IOI (n = 15). (E) Intracellular cytokines secreted by FACS-sorted Tregs from IOI patients (n = 5) were detected after stimulation with IL-33 in the presence of anti-CD3 and anti-CD28 and IL-2. (F) Proliferation of CFSE labeled naïve T cells cocultured with sorted-Tregs under different treatment conditions. (G) Expression of a-SMA in OFs cocultured with Tregs under different treatment conditions. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: In some experiments of CFSE assay and cytokine measurement, Tregs were stimulated with recombinant human (rh)IL-33 (20 ng/mL) (R&D Systems) and human anti-ST2 mAb (R&D Systems), which competitively bound to the IL-33 receptor (ST2).

Techniques: Activation Assay, Clinical Proteomics, Luminex, Expressing, Labeling

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: An imbalance of the IL-33/ST2-AXL-efferocytosis axis induces pregnancy loss through metabolic reprogramming of decidual macrophages

doi: 10.1007/s00018-022-04197-2

Figure Lengend Snippet: sST2 is increased in the serum of patients with unexplained RPL, accompanied by increased apoptotic cells, enhanced efferocytosis and a mitochondrial bias during efferocytosis. Serum samples from 10 patients with normal pregnancies and 12 patients with unexplained RPL were collected to determine the levels of IL-33 (A) and sST2 (B) by ELISAs. The expression of IL-33 (C) and ST2 (D) in dMΦs from the patients with normal pregnancies and unexplained RPL was detected by flow cytometry. (E) The number of apoptotic cells at the maternal–fetal interface of the patients with normal pregnancies and unexplained RPL tested by TUNEL staining. (F) The efferocytosis ability of dMΦs from the patients with normal pregnancies and unexplained RPL. (G) The oxidative phosphorylation (OXPHOS) level of efferocytosis-related metabolism when normal (green solid line) and RPL (red solid line) dMΦs were cocultured with or without AC (indicated by the green dashed and red dashed line, respectively). (H) The glycolytic level of efferocytosis-related metabolism when normal (green solid line) and RPL (red solid line) dMΦs were cocultured with or without AC (indicated by the green dashed and red dashed line, respectively). All experiments were performed in triplicate. The data were analyzed by unpaired Student’s two-tailed t test for comparison of two groups and by two-way ANOVA followed by Tukey’s post-hoc test for multiple groups. The data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns no statistically significant difference

Article Snippet: The fluorescently labeled antibodies used in human cells were as follows: CD14-APC-CY7 (Cat# 301820), CD80-PE (Cat# 305412), CD86-APC (Cat# 305208), CD163-BV421 (Cat# 333612), and CD206-PE-CY7 (Cat# 321124), all obtained from BioLegend; and ST2-PE (Cat# FAB5231P), IL-33-APC (Cat# IC3625A), and AXL-PE (Cat# FAB154P), all purchased from R&D Systems.

Techniques: Expressing, Flow Cytometry, TUNEL Assay, Staining, Phospho-proteomics, Two Tailed Test, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: An imbalance of the IL-33/ST2-AXL-efferocytosis axis induces pregnancy loss through metabolic reprogramming of decidual macrophages

doi: 10.1007/s00018-022-04197-2

Figure Lengend Snippet: Dysfunction of the IL-33/ST2 axis promotes DSC apoptosis and dMΦ/THP-1 efferocytosis and leads to an OXPHOS bias during efferocytosis. (A) The impact of IL-33 (2 ng/ml, 48 h) and sST2 (200 ng/ml, 48 h) on the apoptosis rate of normal DSCs. (B) The impact of sST2 (200 ng/ml, 48 h) on the efferocytosis of normal dMΦs. (C) The mitochondrial membrane potential (mtΔΨ) of the normal and RPL dMΦs conducting efferocytosis. (D) The impact of sST2 (200 ng/ml, 48 h) on the efferocytosis of THP-1 cells. (E) The impact of IL-33 (2 ng/ml, 48 h) and sST2 (200 ng/ml, 48 h) on the mtΔΨ of THP-1 processing efferocytosis. (F) The OXPHOS level of efferocytosis-related metabolism in normal dMΦs affected by IL-33 (2 ng/ml, 48 h) and sST2 (200 ng/ml, 48 h). (G) The glycolytic level of efferocytosis-related metabolism in normal dMΦs affected by IL-33 (2 ng/ml, 48 h) and sST2 (200 ng/ml, 48 h). The effect of IL-33 (2 ng/ml, 48 h) and sST2 (200 ng/ml, 48 h) on the OXPHOS level (H) and glycolytic level (I) of the THP-1 cell line processing efferocytosis. All experiments were performed in triplicate. The data were analyzed by two-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. The data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: The fluorescently labeled antibodies used in human cells were as follows: CD14-APC-CY7 (Cat# 301820), CD80-PE (Cat# 305412), CD86-APC (Cat# 305208), CD163-BV421 (Cat# 333612), and CD206-PE-CY7 (Cat# 321124), all obtained from BioLegend; and ST2-PE (Cat# FAB5231P), IL-33-APC (Cat# IC3625A), and AXL-PE (Cat# FAB154P), all purchased from R&D Systems.

Techniques: Membrane

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: An imbalance of the IL-33/ST2-AXL-efferocytosis axis induces pregnancy loss through metabolic reprogramming of decidual macrophages

doi: 10.1007/s00018-022-04197-2

Figure Lengend Snippet: The IL-33/ST2 axis inhibits AXL expression in dMΦs by activating the PI3K/AKT and ERK1/2 signaling pathways. (A) The GO enrichment analysis diagram of the genes with upregulated expression in ST2-OE THP-1 cells treated with IL-33 for 48 h and the NC group. The effects of the P38 inhibitor (B), JNK inhibitor (C), ERK inhibitor (D), PI3K inhibitor (E), and AKT inhibitor (F) at different concentrations on the AXL expression of normal dMΦs. (G) The impact of ERK, PI3K, and AKT inhibitors on the efferocytosis efficiency of normal dMΦs. All inhibitors were cocultured with CD14+ dMΦs for 24 h, and during the following 48 h, 2 ng/ml IL-33 was added in the presence of inhibitors. (H) Normal dMΦs were treated with IL-33 or sST2 for 48 h, PI3K, AKT and ERK phosphorylation was analyzed by flow cytometry (n = 5), and normal and RPL dMΦs without treatment served as controls. Each experiment was performed at least three times. The data were analyzed by one-way ANOVA with Tukey’s post-hoc test for multiple comparisons. The data are presented as the mean ± SD after analysis with unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns no statistically significant difference

Article Snippet: The fluorescently labeled antibodies used in human cells were as follows: CD14-APC-CY7 (Cat# 301820), CD80-PE (Cat# 305412), CD86-APC (Cat# 305208), CD163-BV421 (Cat# 333612), and CD206-PE-CY7 (Cat# 321124), all obtained from BioLegend; and ST2-PE (Cat# FAB5231P), IL-33-APC (Cat# IC3625A), and AXL-PE (Cat# FAB154P), all purchased from R&D Systems.

Techniques: Expressing, Protein-Protein interactions, Phospho-proteomics, Flow Cytometry

Journal: Experimental & Molecular Medicine

Article Title: A neutrophil elastase-generated mature form of IL-33 is a potent regulator of endothelial cell activation and proliferative retinopathy

doi: 10.1038/s12276-024-01279-y

Figure Lengend Snippet: a Quiescent HRMVEC monolayers were treated with IL-33 95–270 , IL-33 99–270 , IL-33 109–270 , or IL-33 112–270 for 5 min, and cell extracts were prepared. Equal amounts of cell extracts were immunoprecipitated (IP) with an anti-ST2 antibody, and the immunocomplexes were analyzed by Western blotting (WB) with anti-phosphotyrosine (pTyr) and anti-phospho serine/threonine (pSer/Thr) antibodies. The blot was reprobed with anti-ST2 antibodies for lane loading control. b , d Quiescent HRMVEC monolayers were treated with IL-33 95–270 , IL-33 99–270 , IL-33 109–270 , or IL-33 112–270 for 30 min, and cell extracts were prepared and analyzed by Western blotting for the indicated proteins. c Quiescent HRMVEC monolayers were treated with IL-33 95–270 , IL-33 99–270 , IL-33 109–270 , or IL-33 112–270 for 4 h, and cell extracts were prepared and analyzed by Western blotting for the indicated proteins, followed by normalization to β-tubulin. The bar graphs show the quantitative analysis of 3 independent experiments, expressed as the mean ± SD. * p < 0.05 vs. control.

Article Snippet: Anti-ST2 (AF523-SP, dilution 1:500) and anti-IL-33 (AF3626, dilution 1:1000) antibodies and recombinant IL-33 (3625-IL-010/CF) protein were purchased from R&D Systems (Minneapolis, MN).

Techniques: Immunoprecipitation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Acid Stripping of Surface IgE Antibodies Bound to FcεRI Is Unsuitable for the Functional Assays That Require Long-Term Culture of Basophils and Entire Removal of Surface IgE

doi: 10.3390/ijms21020510

Figure Lengend Snippet: Response of the phosphate buffered saline (PBS)-treated human peripheral blood basophils to IL-3 stimulation. Basophils were incubated on ice for 5 min with PBS. Cells were then washed and cultured in serum-free X-VIVO 15 medium (0.1 × 10 6 cells/well per 200 µL) in 96-well U-bottomed plate along with IL-3 (100 ng/0.5 million cells) for 24 h. ( A ) The forward and side scatter plot of the basophils immediately following PBS treatment and following 24-h culture in the presence of IL-3. ( B ) Histogram overlays showing the expression of basophil activation markers CD69, ST2 and FcεRI in PBS-treated basophils cultured for 24 h in IL-3. Representative data from four donors are presented.

Article Snippet: Basophils were analyzed for the expression of FcεRIa (FcεRIa-FITC, clone CRA-1, Miltenyi Biotec, Paris, France), CD69 (CD69-APC/Cy7, Clone FN50, BD Biosciences, Le Pont de Claix, France), CD123 (CD123-BV421, clone 9F5, BD Biosciences), ST2 (ST2/IL-33R-PE polyclonal goat IgG, R&D Systems, Lille, France) and surface intensity of IgE (anti-IgE–APC, clone MB10-5C4, Miltenyi Biotec) by using LSR II flow cytometer (BD Biosciences) and data were analyzed by BD FACSDiva v8.0.1 (BD Biosciences) and FlowJo v10 softwares (FlowJo, LLC, Ashland, USA).

Techniques: Saline, Incubation, Cell Culture, Expressing, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Acid Stripping of Surface IgE Antibodies Bound to FcεRI Is Unsuitable for the Functional Assays That Require Long-Term Culture of Basophils and Entire Removal of Surface IgE

doi: 10.3390/ijms21020510

Figure Lengend Snippet: Response of the acetic acid buffer (pH 4)-treated human basophils to IL-3 stimulation. Basophils were incubated on ice for 5 min either with phosphate buffered saline (PBS) or ice-cold acetic acid buffer pH 4 (AA pH 4). Cells were then washed and cultured in serum-free X-VIVO 15 medium (0.1 × 10 6 cells/well per 200 µL) in 96-well U-bottomed plate along with IL-3 (100 ng/0.5 million cells) for 24 h. ( A ) The forward and side scatter plot of AA pH 4-treated basophils following 24-h culture in IL-3. ( B ) The yield of basophils (mean ± SD, n = 4 donors) after 24 h culture of basophils in IL-3. The values were calculated based on the percentage of cells in the P1 gate of forward and side scatter plot. ( C ) Histogram overlays showing the expression of basophil activation markers CD69, ST2 and FcεRI in AA pH 4-treated cells after 24 h culture in IL-3. Representative data from four donors are presented. ( D ) The expression (mean ± SD, n = 4 donors) of various activation markers (% positive cells or median fluorescence intensity, MFI) after 24 h culture of basophils in IL-3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant; two-sided Students t-test (panel B) or one-way ANOVA with Tukey’s multiple comparison test (panel D).

Article Snippet: Basophils were analyzed for the expression of FcεRIa (FcεRIa-FITC, clone CRA-1, Miltenyi Biotec, Paris, France), CD69 (CD69-APC/Cy7, Clone FN50, BD Biosciences, Le Pont de Claix, France), CD123 (CD123-BV421, clone 9F5, BD Biosciences), ST2 (ST2/IL-33R-PE polyclonal goat IgG, R&D Systems, Lille, France) and surface intensity of IgE (anti-IgE–APC, clone MB10-5C4, Miltenyi Biotec) by using LSR II flow cytometer (BD Biosciences) and data were analyzed by BD FACSDiva v8.0.1 (BD Biosciences) and FlowJo v10 softwares (FlowJo, LLC, Ashland, USA).

Techniques: Incubation, Saline, Cell Culture, Expressing, Activation Assay, Fluorescence, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Acid Stripping of Surface IgE Antibodies Bound to FcεRI Is Unsuitable for the Functional Assays That Require Long-Term Culture of Basophils and Entire Removal of Surface IgE

doi: 10.3390/ijms21020510

Figure Lengend Snippet: Lactic acid treatment does not affect viability, yield and IL-3 induced activation of basophils, but removes only a small fraction of the cell surface bound IgE. Basophils were incubated on ice either with phosphate buffered saline (PBS) or ice-cold lactic acid buffer pH 3.9 (LA). Cells were then washed and cultured in serum-free X-VIVO 15 medium (0.1 × 10 6 cells/well per 200 µL) in 96-well U-bottomed plate along with IL-3 (100 ng/0.5 million cells) for 24 h. ( A ) The viability of cells immediately following LA treatment as analyzed by fixable viable dye staining. ( B ) The forward and side scatter plot of LA-treated basophils following 24-h culture in IL-3. ( C ) The expression of basophil activation markers ST2, FcεRI and CD69 in LA-treated cells after 24 h culture in IL-3 (mean ± SD, n = 4 experiments from two donors). ( D ) Efficacy of stripping of basophil surface-bound IgE by LA as analyzed by surface staining of IgE and analyses by flow cytometry. The intensity of IgE on the surface of basophils was represented by MFI values (Median fluorescence intensity). ** p < 0.01; *** p < 0.001; **** p < 0.0001; one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: Basophils were analyzed for the expression of FcεRIa (FcεRIa-FITC, clone CRA-1, Miltenyi Biotec, Paris, France), CD69 (CD69-APC/Cy7, Clone FN50, BD Biosciences, Le Pont de Claix, France), CD123 (CD123-BV421, clone 9F5, BD Biosciences), ST2 (ST2/IL-33R-PE polyclonal goat IgG, R&D Systems, Lille, France) and surface intensity of IgE (anti-IgE–APC, clone MB10-5C4, Miltenyi Biotec) by using LSR II flow cytometer (BD Biosciences) and data were analyzed by BD FACSDiva v8.0.1 (BD Biosciences) and FlowJo v10 softwares (FlowJo, LLC, Ashland, USA).

Techniques: Activation Assay, Incubation, Saline, Cell Culture, Staining, Expressing, Stripping Membranes, Flow Cytometry, Fluorescence, Comparison