Journal: BMC pharmacology & toxicology

Article Title: Blocking group 2 innate lymphoid cell activation and macrophage M2 polarization: potential therapeutic mechanisms in ovalbumin-induced allergic asthma by calycosin.

doi: 10.1186/s40360-024-00751-9

Figure Lengend Snippet: Fig. 2 Calycosin inhibited group 2 innate lymphoid cell (ILC2) activation in OVA-induced asthmatic mice. (A) The levels of interleukin (IL)-4, (B) IL-5 and (C) IL-13 in the BALF and lung tissues were measured using ELISA and RT-qPCR. (D) ELISA was used to determin the contents of IL-33 in the BALF. (E) Suppression tumorigenicity 2 (ST2) levels in the lung tissues was examined by western blot. The blots were cut prior to hybridization with antibodies during blotting. (F) Immunohistochemistry (IHC) staining was used to detect the expression ST2 and IL-25R. (G) The co-localization of ST2 and CD45 was detected by immunofluorescence (IF). Scale bar = 50 μm. n = 6 for each group. * P< 0.05, **P < 0.01, ***P < 0.001, compared with the sham group; #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the OVA group. “Ca” represents calycosin

Article Snippet: The sections were incubated with ST2 antibody (1:50; 11920- 1-AP, ProteinTech, Wuhan, China) at 4°C overnight and subjected to HRP-labeled goat anti-rabbit IgG (1:500; # 31460, ThermoFisher Scientific, USA) at 37°C for 1 h. Additionally, IL-25R expression was detected in the same way.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Hybridization, Immunohistochemistry, Expressing, Immunofluorescence

Journal: BMC pharmacology & toxicology

Article Title: Blocking group 2 innate lymphoid cell activation and macrophage M2 polarization: potential therapeutic mechanisms in ovalbumin-induced allergic asthma by calycosin.

doi: 10.1186/s40360-024-00751-9

Figure Lengend Snippet: Fig. 4 ILC2 activation induced by IL-33 was restrained by calycosin in ILC2s. (A) The ILC2 cell viability was examined by CCK-8. (B) Western blot showed the expression of ST2 in ILC2s. The blots were cut prior to hybridization with antibodies during blotting. (C) The secretion of IL-4, (D) IL-5 and (E) IL-13 in ILC2s were detected by ELISA assay. n = 3 for each group. **P < 0.01,***P < 0.001, compared with the control group; #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the IL-33 group. “Ca” represents calycosin

Article Snippet: The sections were incubated with ST2 antibody (1:50; 11920- 1-AP, ProteinTech, Wuhan, China) at 4°C overnight and subjected to HRP-labeled goat anti-rabbit IgG (1:500; # 31460, ThermoFisher Scientific, USA) at 37°C for 1 h. Additionally, IL-25R expression was detected in the same way.

Techniques: Activation Assay, CCK-8 Assay, Western Blot, Expressing, Hybridization, Enzyme-linked Immunosorbent Assay, Control

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: iScience

Article Title: Direct activation of toll-like receptor 4 signaling in group 2 innate lymphoid cells contributes to inflammatory responses of allergic diseases

doi: 10.1016/j.isci.2024.111240

Figure Lengend Snippet: LPS activates ILC2s via the TLR4 receptor without the involvement of the IL-33-ST2 pathway (A) Heatmap showing the normalized mRNA levels of all 10 human TLRs in human ILC2s from two donors. (B) FACS staining of TLR4 or CD14 proteins on the surface of human ILC2s. Light microscopic images showing the growth of human ILC2s treated with increasing doses of CRX-527, a lipid A analog, or its solvent DMSO (C). Scale bar, 100 μm. (D) The number of human ILC2s was quantified by FACS analysis. IL-5 (E) and IL-13 (F) from human ILC2s were examined by ELISA. Light microscopic images showing human ILC2s exposed to increasing doses of LPS-RS (G). Scale bar, 100 μm. (H) The number of human ILC2s was quantified using FACS. ELISA was used to measure the production of IL-5 (I) and IL-13 (J) from human ILC2s (unpaired t -test, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). Human ILC2s were treated by increasing doses of recombinant protein IL1RL1, and light microscopy (K) was used to observe their growth. Scale bar, 100 μm. (L) FACS analysis showing the number of human ILC2s. ELISA measuring the production of IL-5 (M) and IL-13 (N) by human ILC2s (two-way ANOVA, ∗∗∗∗ p < 0.0001). The data are representative from 2 experiments.

Article Snippet: Human IL1RL1/ST2 protein (isoform a, His Tag; 13034-H08H) was purchased from Sino Biological.

Techniques: Staining, Solvent, Enzyme-linked Immunosorbent Assay, Recombinant, Light Microscopy

Journal: iScience

Article Title: Direct activation of toll-like receptor 4 signaling in group 2 innate lymphoid cells contributes to inflammatory responses of allergic diseases

doi: 10.1016/j.isci.2024.111240

Figure Lengend Snippet:

Article Snippet: Human IL1RL1/ST2 protein (isoform a, His Tag; 13034-H08H) was purchased from Sino Biological.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Generated, Sequencing, Software

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Ras (G12V) causes the expression of ST2 gene products in NIH-3T3 murine fibroblasts. (A) NIH-3T3 cells were infected with the empty retrovirus (CTR) or retrovirus harboring Ras (G12V), and infected cells were selected by the treatment with puromycin (7.5 μg/ml) for 72 h. Then, total RNA was extracted from infected cells, and the expression levels of ST2 gene products were analyzed by quantitative PCR. In the graph, error bars = S.D. (n = 3, *P < 0.005). (B) Conditioned medium and whole cell lysates were concentrated, and treated with N-glycosidase F. Then, ST2 and ST2L proteins induced by Ras (G12V) were detected by immunoblot analysis with anti-mouse ST2 antibody, and β-actin was also stained as a loading control for the immunoblot analysis. The intensities of bands were quantified, and shown in the graphs. In the graph, error bars = S.D. (n = 3, *P<0.005). Original blots are shown in supplementary file named as “Original blots for Ras & ST2”.

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Western Blot, Staining, Control

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Enforced expression of ST2 gene products in NIH-3T3 cells transformed by oncogenic Ras. (A) We diluted retroviruses to forcibly express the various amount Ras (G12V) by adjusting the amounts of retroviruses harboring gene of Ras (G12V), such as 50- (lane 2, 6, and 10), 15- (lane 3, 7, and 11), and 5-fold (lane 4, 8, and 12) dilution, and infected cells were selected by the treatment with puromycin for 72 h. To co-express ST2-FLAG and ST2L-FLAG, we then infected the retroviruses harboring ST2-FLAG and ST2L-FLAG, respectively. After selection with puromycin, cells were treated with serum starvation for 24 h. Then, whole cell lysates (WCL) and conditioned media (CM) were prepared, and analyzed by immunoblotting with the primary antibodies such as anti-FLAG (M2), H-Ras, cyclin D1, Rb, phosphorylated Rb (Ser-807/811), and β-actin antibodies, respectively. To detect secreted ST2, the culture supernatant (conditioned media: CM) was also analyzed. The positions of bands for ST2L and ST2 are indicated. Original blots are shown in supplementary file named as “Original blots for Ras & ST2”. (B) The intensity of the expression of total Rb protein, the ratio between Rb phosphorylation and total Rb, and the expression of cyclin D1 protein were quantitated, calculated, and then results were shown in graphs. In the graph, error bars = S.D. (n = 3, *P<0.005).

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Expressing, Transformation Assay, Infection, Selection, Western Blot, Phospho-proteomics

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Effect of enforced expression of ST2 gene products on the expression of Ras protein and cyclin D1 mRNA. (A) In the cells analyzed in , the intensity of the expression of H-Ras protein, which were normalized with the expression of β-actin, and then results were shown in graph. In the graph, error bars = S.D. (n = 3). (B) The expressional ratio between cyclin D1 and H-Ras was calculated, and then results were shown in graph. In the graph, error bars = S.D. (n = 3). (C) Infected cells were utilized for the total RNA extraction, and analyzed by qPCR for evaluating the effect of Ras (G12V) and ST2 gene products on the expression of cyclin D1 mRNA. The results were shown in graph. In the graph, error bars = S.D. (n = 3, *P < 0.005). (D) In the cells analyzed in , infected cells were seeded onto soft agar at 1 × 10 4 cells per 35 mm-diameter dishes, and grown for 2–3 weeks. The colonies were visualized by the treatment with MTT, and the results were shown in graph (magnification: 4 ×). In the graph, error bars = S.D. (n = 3, *P<0.005).

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Expressing, Infection, RNA Extraction

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Enforced expression of ST2 gene products accelerates cellular transformation induced by Ras (G12V). (A) NIH-3T3 cells were infected with the indicated combination of retroviruses harboring Ras (G12V), ST2-FLAG, and ST2L-FLAG, and infected cells were selected by the treatment with puromycin for 72 h. Then, a total of 3 × 10 3 infected cells were mixed with 1 × 10 5 uninfected cells and seeded onto 60 mm-diameter dishes. Two weeks later, transformed foci were stained with Giemsa, and then photographed at 4 × magnification. (B) The numbers of foci shown in (A) were counted, and the results were shown in the graph. In the graph, error bars = S.D. (n = 3, *P < 0.01). (C) Infected NIH-3T3 cells were seeded onto soft agar at 1 × 10 4 cells per 35-mm dish, and grown for 2–3 weeks. Grown colonies were photographed at 4 × magnification, and the numbers of colonies were counted. (D) The number of colonies of transformed NIH-3T3 cells counted in (C) is shown in the graph. In the graph, error bars = S.D. (n = 3, *P < 0.005). (E) To evaluate the resistance to anoikis, cells were suspended in DMEM including 1% FBS, and then seeded onto the 24-well plates treated with poly-HEMA. Twenty-hour hours later, cell viability was measured using a WST-1 assay. In the graph, error bars = S.D. (n = 3, *P<0.01).

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Expressing, Transformation Assay, Infection, Staining, WST-1 Assay

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Soluble ST2 accelerated the cellular transformation in paracrine- and autocrine-dependent manner. (A) Then infected NIH-3T3 cells analyzed in were seeded on slide glasses, and fixed with paraformaldehyde. Then, the expression of FLAG-tagged ST2 and ST2L were detected by immuno-staining analysis using anti-FLAG (green). Nucleus was also stained with DAPI (blue). In bottom photographs, scale bars were added (length: 153 μm) (B) Conditioned medium was also collected from the infected NIH-3T3 cells analyzed in (A). Then, concentration of ST2 was analyzed by ELISA. In the graph, error bars = S.D. (n = 3, *P < 0.001). (C) Using conditioned medium from control NIH-3T3 and Ras (G12V)-transformed NIH-3T3 cells, ELISA for IL-33 was also performed. Recombinant IL-33 (rIL-33) was utilized as a positive control. In the graph, error bars = S.D. (n = 3, *P < 0.005). N.D. means “Not detected”. The expressions of Ras (G12V) and intracellular IL-33 were detected by immunoblot analysis. The blot for β-tubulin is a loading control. Original blots are shown in supplementary file named as “Original blots for Ras & ST2”. (D) To test the paracrine- and autocrine-dependent effect of ST2 on cellular transformation, 3 × 10 3 untransformed NIH-3T3 cells or transformed cells provoked by Ras (G12V) were mixed with 1 × 10 5 normal NIH-3T3 (top photograph), NIH-3T3 producing soluble ST2 (middle photograph), or NIH-3T3 cells expressing ST2L (bottom photograph), and then cultured in 60-mm diameter dishes during 2 weeks. Foci were visualized by staining with Giemsa reagent, and then photographed at 4 × magnification. (E) The numbers of foci shown in (D) were counted, and the results were shown in the graph. In the graph, error bars = S.D. (n = 3, *P<0.005).

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Transformation Assay, Infection, Expressing, Immunostaining, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Recombinant, Positive Control, Western Blot, Cell Culture

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Knockdown of the expression of ST2 gene products in normal and transformed NIH-3T3 cells. (A) NIH-3T3 cells were infected with the indicated retroviruses including Ras (G12V), sh-luciferase (sh-Luc), and sh-ST2/ST2L (Referred to as sh-ST2), and infected cells were selected by the treatment with puromycin for 72 h. And then, total RNA was extracted from infected cells, and the expression levels of ST2 gene products were analyzed by quantitative PCR. In the graph, error bars = S.D. (n = 3, *P < 0.005). (B) The reduction of the protein expression of ST2 and ST2L by sh-RNA was detected by immunoblot analysis. Original blots are shown in supplementary file named as “Original blots for Ras & ST2”. (C) The results of (B) were shown in the graphs, respectively. In the graph, error bars = S.D. (n = 3, *P<0.005).

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Knockdown, Expressing, Transformation Assay, Infection, Luciferase, Real-time Polymerase Chain Reaction, Western Blot

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Silencing the expression of ST2 gene products suppresses the cellular transformation induced by Ras (G12V). (A) Infected NIH-3T3 cells were seeded onto soft agar at 1 × 10 4 cells per 35-mm dish, and grown for 2–3 weeks. Grown colonies were photographed at 4 × magnification, and the numbers of colonies were counted. In the labels of photographs, RasV means Ras (G12V) mutant. (B) The number of colonies of transformed NIH-3T3 cells counted in (A) is shown in the graph. In the graph, error bars = S.D. (n = 3, *P < 0.005). N.D. means “Not detected”. (C) Whole cell lysates were prepared from the infected cells and analyzed by immunoblotting with the primary antibodies indicated on the left of the image. Original blots are shown in supplementary file named as “Original blots for Ras & ST2”. (D) The intensity of Rb phosphorylation, total Rb, cyclin D1, and the ratio between phosphorylated Rb and total Rb were quantitated, calculated, and then results were shown in graphs, respectively. In the graph, error bars = S.D. (n = 3, *P<0.005).

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Expressing, Transformation Assay, Infection, Mutagenesis, Western Blot, Phospho-proteomics

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: ST2 gene products may modulate the regulation of the cell cycle . (A) NIH-3T3 cells were infected with the indicated retroviruses. Infected cells were held in G 0 phase by serum starvation for 24 h. Then, the cells were released into the cell cycle by dividing the cells onto new dishes. At the indicated time points, cells were harvested and stained with propidium iodide. Cell suspension was analyzed by flow cytometry. (B) The percentage of cells entering into S-phase was shown in graph. Significant difference of the cell population entering to S-phase at 20 hr after cell cycle release between ST2-knockdowned cells and control cells was statistically determined by the Student's t -test. In the graph, error bars = S.D. (n = 3, *P < 0.005). (C) At indicated time points, cells were harvested and analyzed by immunoblot with primary antibodies, as indicated at the left of the image. Original blots are shown in supplementary file named as “Original blots for Ras & ST2”.

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Infection, Staining, Suspension, Flow Cytometry, Control, Western Blot

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Working hypothesis for the mechanism how ST2 gene products contribute to the tumor growth and progression.

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques:

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