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R&D Systems staining st2 il 33r

(A) A schematic of experimental design. CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 alone, IL2 and IL-33, or anti-CD3/CD28 under Th2 polarizing conditions for five days. (B) Representative flow plot of CD4 T cells immediately following isolation from secondary lymphoid organs of naïve or Hpoly -infected mice. (C) CD4 T cells from secondary lymphoid organs of Hpoly -infected mice were cultured under indicated conditions. Frequencies and numbers of <t>GATA3+ST2+</t> cells were quantified. (D) Representative flow plot of CD4 T cells right after isolation from secondary lymphoid organs of Hpoly-infected 4get mice. (E) CD4 T cells from naïve or Hpoly-infected mice were cultured with IL2 and IL-33. (F) CD4 T cells from Hpoly-infected mice were cultured under the indicated conditions. Frequencies and numbers of GATA3+ST2+ cells were quantified after five days. (G) Abundance of amphiregulin in the culture supernatants from (F). (H) Splenocytes from naïve or Hpoly-infected mice cultured with IL2 alone or together with HES. Frequencies and numbers of GATA3+ cells was quantified. The data (B-H) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B-G) or two-way ANOVA with Sidak post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Staining St2 Il 33r, supplied by R&D Systems, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells"

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

Journal: bioRxiv

doi: 10.1101/2025.07.09.663905

Figure Legend Snippet: (A) A schematic of experimental design. CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 alone, IL2 and IL-33, or anti-CD3/CD28 under Th2 polarizing conditions for five days. (B) Representative flow plot of CD4 T cells immediately following isolation from secondary lymphoid organs of naïve or Hpoly -infected mice. (C) CD4 T cells from secondary lymphoid organs of Hpoly -infected mice were cultured under indicated conditions. Frequencies and numbers of GATA3+ST2+ cells were quantified. (D) Representative flow plot of CD4 T cells right after isolation from secondary lymphoid organs of Hpoly-infected 4get mice. (E) CD4 T cells from naïve or Hpoly-infected mice were cultured with IL2 and IL-33. (F) CD4 T cells from Hpoly-infected mice were cultured under the indicated conditions. Frequencies and numbers of GATA3+ST2+ cells were quantified after five days. (G) Abundance of amphiregulin in the culture supernatants from (F). (H) Splenocytes from naïve or Hpoly-infected mice cultured with IL2 alone or together with HES. Frequencies and numbers of GATA3+ cells was quantified. The data (B-H) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B-G) or two-way ANOVA with Sidak post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Techniques Used: Isolation, Infection, Cell Culture

(A) Representative flow plots and quantification of ST2+GATA3 + in live CD45+TCRβ+CD4+FOXP3-T cells from mesenteric adipose tissue of Hpoly -infected mice or after five days of in vitro culture of CD4 T cells isolated from Hpoly-infected mice. Flow plots and quantification of ST2+IL4+(GFP) (B) or CD44+IL4+(GFP+) (C) cells cultured as above. (D) CD4 T cells from naïve or Hpoly -infected mice were cultured as in (A) and IL-33R+GATA3+ Th2 cells were quantified. (E) Representative flow plots and quantification of AREG+ cells within the IL4+(GFP+) population after 5hr restimulation with PMA/Iono in the presence of Brefeldin. The data (A-E) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (A-C, E) or two-way ANOVA with Sidak post hoc test (D).. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, *** p ≤ 0.0001.

Figure Legend Snippet: (A) Representative flow plots and quantification of ST2+GATA3 + in live CD45+TCRβ+CD4+FOXP3-T cells from mesenteric adipose tissue of Hpoly -infected mice or after five days of in vitro culture of CD4 T cells isolated from Hpoly-infected mice. Flow plots and quantification of ST2+IL4+(GFP) (B) or CD44+IL4+(GFP+) (C) cells cultured as above. (D) CD4 T cells from naïve or Hpoly -infected mice were cultured as in (A) and IL-33R+GATA3+ Th2 cells were quantified. (E) Representative flow plots and quantification of AREG+ cells within the IL4+(GFP+) population after 5hr restimulation with PMA/Iono in the presence of Brefeldin. The data (A-E) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (A-C, E) or two-way ANOVA with Sidak post hoc test (D).. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, *** p ≤ 0.0001.

Techniques Used: Infection, In Vitro, Isolation, Cell Culture

(A) CD44+IL4+ (GFP)+ early Th2 cells, CD44+IL4-(GPF-) activated cells, and naïve CD44-GFP-cells were FACS-isolated from 4get mice infected with Hpoly and cultured with IL2 and IL-33 for five days. Frequencies of GFP+ and GFP+ST2+ cells (B) as well as ST2 gMFI were quantified (C). The data (A-C) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B) or paired T-test (C). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure Legend Snippet: (A) CD44+IL4+ (GFP)+ early Th2 cells, CD44+IL4-(GPF-) activated cells, and naïve CD44-GFP-cells were FACS-isolated from 4get mice infected with Hpoly and cultured with IL2 and IL-33 for five days. Frequencies of GFP+ and GFP+ST2+ cells (B) as well as ST2 gMFI were quantified (C). The data (A-C) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B) or paired T-test (C). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Techniques Used: Isolation, Infection, Cell Culture

RNAseq was performed on CD44+IL4+(GFP+) Th2 cells isolated from spleens and lymph nodes of Hpoly -infected mice or after five-day culture under indicated conditions. (A) Principal component analysis of RNAseq data. (B) Heatmap of hierarchical clustering of differentially expressed genes. (C) Top Hallmark pathways from GSEA for IL-33 v IL2 comparison. (D) Flow plots and quantification of pS6 expression in CD44+IL4(GFP+) Th2 cells. (E) CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 and IL-33 in the presence of rapamycin. Frequencies and numbers of GATA3+ST2+ Th2 cells were quantified. (F) Levels of indicated cytokines after overnight culture in fresh media containing rapamycin. The data (D, E, F) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (D, E) or paired T-test (F). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure Legend Snippet: RNAseq was performed on CD44+IL4+(GFP+) Th2 cells isolated from spleens and lymph nodes of Hpoly -infected mice or after five-day culture under indicated conditions. (A) Principal component analysis of RNAseq data. (B) Heatmap of hierarchical clustering of differentially expressed genes. (C) Top Hallmark pathways from GSEA for IL-33 v IL2 comparison. (D) Flow plots and quantification of pS6 expression in CD44+IL4(GFP+) Th2 cells. (E) CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 and IL-33 in the presence of rapamycin. Frequencies and numbers of GATA3+ST2+ Th2 cells were quantified. (F) Levels of indicated cytokines after overnight culture in fresh media containing rapamycin. The data (D, E, F) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (D, E) or paired T-test (F). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Techniques Used: Isolation, Infection, Comparison, Expressing, Cell Culture

(A) Top Hallmark pathways from GSEA for IL-33 v IL2 comparison. (B) CD4 T cells were isolated from Hpoly -infected mice and cultured anti-CD3/CD28 in the presence of rapamycin. The mean fluorescence intensity of GATA3 and cell numbers were quantified. (C) Levels of indicated cytokines after overnight culture of anti-CD3/CD28-stimulated cells in fresh media containing rapamycin. (D) Quantification of results from SCENITH assay in GATA3+ Th2 cells. (E) Representative flow plot showing the expression of TSLPR and ST2 after five days of in vitro culture of CD4 T cells were isolated from Hpoly -infected mice. (F) Representative flow plot and quantification of mitotracker green and mitotracker deep red staining after five days of culture. The data (B-F) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B, E, F), or paired T-test (C). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure Legend Snippet: (A) Top Hallmark pathways from GSEA for IL-33 v IL2 comparison. (B) CD4 T cells were isolated from Hpoly -infected mice and cultured anti-CD3/CD28 in the presence of rapamycin. The mean fluorescence intensity of GATA3 and cell numbers were quantified. (C) Levels of indicated cytokines after overnight culture of anti-CD3/CD28-stimulated cells in fresh media containing rapamycin. (D) Quantification of results from SCENITH assay in GATA3+ Th2 cells. (E) Representative flow plot showing the expression of TSLPR and ST2 after five days of in vitro culture of CD4 T cells were isolated from Hpoly -infected mice. (F) Representative flow plot and quantification of mitotracker green and mitotracker deep red staining after five days of culture. The data (B-F) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B, E, F), or paired T-test (C). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Techniques Used: Comparison, Isolation, Infection, Cell Culture, Fluorescence, Expressing, In Vitro, Staining

(A) Heatmap representing the abundance of the top 100 detected metabolites. (B) CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 and IL-33 for five days. The intracellular abundance of indicated amino acids in naïve and CD4+CD44+ activated cells was determined via LC/MS. (C) Heatmap depicting the expression of arginine transporters genes related in RNAseq data from . (D) CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 and IL-33 for five days in amino acid drop-out media. Frequencies and numbers of GATA3+ST2+ Th2 cells were quantified. (E) Representative histograms and quantification of CTV dilution of CD4 T cells cultured as in D. (F) After five days of culture with IL2 and IL-33 in control or arginine-depleted media, cells were restimulated with PMA/Iono and amphiregulin was quantified. (G) Representative histograms and quantification of ps6 levels in pS6-positive cells at day 1 of culture. The data (D-G) are representative of at least two independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (D, E), paired T-test (F), or two-way ANOVA with Sidak post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure Legend Snippet: (A) Heatmap representing the abundance of the top 100 detected metabolites. (B) CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 and IL-33 for five days. The intracellular abundance of indicated amino acids in naïve and CD4+CD44+ activated cells was determined via LC/MS. (C) Heatmap depicting the expression of arginine transporters genes related in RNAseq data from . (D) CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 and IL-33 for five days in amino acid drop-out media. Frequencies and numbers of GATA3+ST2+ Th2 cells were quantified. (E) Representative histograms and quantification of CTV dilution of CD4 T cells cultured as in D. (F) After five days of culture with IL2 and IL-33 in control or arginine-depleted media, cells were restimulated with PMA/Iono and amphiregulin was quantified. (G) Representative histograms and quantification of ps6 levels in pS6-positive cells at day 1 of culture. The data (D-G) are representative of at least two independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (D, E), paired T-test (F), or two-way ANOVA with Sidak post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Techniques Used: Isolation, Infection, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Expressing, Control

(A) Expresssion of Th2 cell related genes in scRNAseq dataset of T cells isolated from mAT of Hpoly -infected mice identifying cluster of GATA3+ST2+ Th2 cells (circled). CD4 T cells from Hpoly-infected mice were stimulated with anti-CD3/CD28 and treated with DFMO (B) or cultured in arginine-depleted media (C). Mean fluorescence intestity of GATA3 and cells numbers were quantified. The data (B,C) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B, C). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure Legend Snippet: (A) Expresssion of Th2 cell related genes in scRNAseq dataset of T cells isolated from mAT of Hpoly -infected mice identifying cluster of GATA3+ST2+ Th2 cells (circled). CD4 T cells from Hpoly-infected mice were stimulated with anti-CD3/CD28 and treated with DFMO (B) or cultured in arginine-depleted media (C). Mean fluorescence intestity of GATA3 and cells numbers were quantified. The data (B,C) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B, C). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Techniques Used: Isolation, Infection, Cell Culture, Fluorescence

Image Search Results

Journal: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: (A) A schematic of experimental design. CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 alone, IL2 and IL-33, or anti-CD3/CD28 under Th2 polarizing conditions for five days. (B) Representative flow plot of CD4 T cells immediately following isolation from secondary lymphoid organs of naïve or Hpoly -infected mice. (C) CD4 T cells from secondary lymphoid organs of Hpoly -infected mice were cultured under indicated conditions. Frequencies and numbers of GATA3+ST2+ cells were quantified. (D) Representative flow plot of CD4 T cells right after isolation from secondary lymphoid organs of Hpoly-infected 4get mice. (E) CD4 T cells from naïve or Hpoly-infected mice were cultured with IL2 and IL-33. (F) CD4 T cells from Hpoly-infected mice were cultured under the indicated conditions. Frequencies and numbers of GATA3+ST2+ cells were quantified after five days. (G) Abundance of amphiregulin in the culture supernatants from (F). (H) Splenocytes from naïve or Hpoly-infected mice cultured with IL2 alone or together with HES. Frequencies and numbers of GATA3+ cells was quantified. The data (B-H) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B-G) or two-way ANOVA with Sidak post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Isolation, Infection, Cell Culture

Journal: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: (A) Representative flow plots and quantification of ST2+GATA3 + in live CD45+TCRβ+CD4+FOXP3-T cells from mesenteric adipose tissue of Hpoly -infected mice or after five days of in vitro culture of CD4 T cells isolated from Hpoly-infected mice. Flow plots and quantification of ST2+IL4+(GFP) (B) or CD44+IL4+(GFP+) (C) cells cultured as above. (D) CD4 T cells from naïve or Hpoly -infected mice were cultured as in (A) and IL-33R+GATA3+ Th2 cells were quantified. (E) Representative flow plots and quantification of AREG+ cells within the IL4+(GFP+) population after 5hr restimulation with PMA/Iono in the presence of Brefeldin. The data (A-E) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (A-C, E) or two-way ANOVA with Sidak post hoc test (D).. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, *** p ≤ 0.0001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Infection, In Vitro, Isolation, Cell Culture

Journal: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: (A) CD44+IL4+ (GFP)+ early Th2 cells, CD44+IL4-(GPF-) activated cells, and naïve CD44-GFP-cells were FACS-isolated from 4get mice infected with Hpoly and cultured with IL2 and IL-33 for five days. Frequencies of GFP+ and GFP+ST2+ cells (B) as well as ST2 gMFI were quantified (C). The data (A-C) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B) or paired T-test (C). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Isolation, Infection, Cell Culture

Journal: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: RNAseq was performed on CD44+IL4+(GFP+) Th2 cells isolated from spleens and lymph nodes of Hpoly -infected mice or after five-day culture under indicated conditions. (A) Principal component analysis of RNAseq data. (B) Heatmap of hierarchical clustering of differentially expressed genes. (C) Top Hallmark pathways from GSEA for IL-33 v IL2 comparison. (D) Flow plots and quantification of pS6 expression in CD44+IL4(GFP+) Th2 cells. (E) CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 and IL-33 in the presence of rapamycin. Frequencies and numbers of GATA3+ST2+ Th2 cells were quantified. (F) Levels of indicated cytokines after overnight culture in fresh media containing rapamycin. The data (D, E, F) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (D, E) or paired T-test (F). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Isolation, Infection, Comparison, Expressing, Cell Culture

Journal: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: (A) Top Hallmark pathways from GSEA for IL-33 v IL2 comparison. (B) CD4 T cells were isolated from Hpoly -infected mice and cultured anti-CD3/CD28 in the presence of rapamycin. The mean fluorescence intensity of GATA3 and cell numbers were quantified. (C) Levels of indicated cytokines after overnight culture of anti-CD3/CD28-stimulated cells in fresh media containing rapamycin. (D) Quantification of results from SCENITH assay in GATA3+ Th2 cells. (E) Representative flow plot showing the expression of TSLPR and ST2 after five days of in vitro culture of CD4 T cells were isolated from Hpoly -infected mice. (F) Representative flow plot and quantification of mitotracker green and mitotracker deep red staining after five days of culture. The data (B-F) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B, E, F), or paired T-test (C). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Comparison, Isolation, Infection, Cell Culture, Fluorescence, Expressing, In Vitro, Staining

Journal: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: (A) Heatmap representing the abundance of the top 100 detected metabolites. (B) CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 and IL-33 for five days. The intracellular abundance of indicated amino acids in naïve and CD4+CD44+ activated cells was determined via LC/MS. (C) Heatmap depicting the expression of arginine transporters genes related in RNAseq data from . (D) CD4 T cells were isolated from Hpoly -infected mice and cultured with IL2 and IL-33 for five days in amino acid drop-out media. Frequencies and numbers of GATA3+ST2+ Th2 cells were quantified. (E) Representative histograms and quantification of CTV dilution of CD4 T cells cultured as in D. (F) After five days of culture with IL2 and IL-33 in control or arginine-depleted media, cells were restimulated with PMA/Iono and amphiregulin was quantified. (G) Representative histograms and quantification of ps6 levels in pS6-positive cells at day 1 of culture. The data (D-G) are representative of at least two independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (D, E), paired T-test (F), or two-way ANOVA with Sidak post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Isolation, Infection, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Expressing, Control

Journal: bioRxiv

Article Title: IL-33 promotes transcriptional and metabolic adaptations of tissue-resident Th2 cells

doi: 10.1101/2025.07.09.663905

Figure Lengend Snippet: (A) Expresssion of Th2 cell related genes in scRNAseq dataset of T cells isolated from mAT of Hpoly -infected mice identifying cluster of GATA3+ST2+ Th2 cells (circled). CD4 T cells from Hpoly-infected mice were stimulated with anti-CD3/CD28 and treated with DFMO (B) or cultured in arginine-depleted media (C). Mean fluorescence intestity of GATA3 and cells numbers were quantified. The data (B,C) are representative of at least two independent experiments. Symbols in the quantified data represent independent biological replicates. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (B, C). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Additionally, biotinylated antibodies were used for the staining ST2 (IL-33R) (MDB, clone DJ8) and amphiregulin (R&D Systems, #BAF989).

Techniques: Isolation, Infection, Cell Culture, Fluorescence

(A) Histogram presentation of ST2 expression in FcεRIα+ and FcεRIαneg macrophages in SCC. (B) Flow cytometry analysis of FcεRIα and ST2 expression in CSF1- and IL-33–induced F4/80+ macrophages from bone marrow (BM)–derived cells (see fig. S5B). (C) qPCR analysis of CSF1-induced, IL-33–induced, and CSF1-induced→IL-4–treated macrophages in vitro (n = 3). Data are shown as relative mean expression with SD. (D) qPCR analysis of IL-33–induced macrophages treated with a MEK inhibitor (U0126, 0.5 –M), a JNK inhibitor (SP600125, 0.5 –M), a p38 inhibitor (SB202190, 0.5 –M), or an NF-κB inhibitor (BAY11-7082, 1 μM) during macrophage differentiation (n = 3). Data are shown as relative mean expression with SD and were analyzed with unpaired t test, **P = 0.0022. (E) qPCR analysis of IL-33–induced and IL-4–activated macrophages treated with the NF-κB inhibitor (n = 3). Data are shown as relative mean expression with SD and were analyzed with unpaired t test, ***P < 0.001. (F and G) qPCR analysis of IL-33–induced macrophages from ex vivo expanded hematopoietic progenitor cells that were LV transduced with scramble control, (F) Rela (NF-κB p65) shRNA, or (G) Fcer1a shRNA (see fig. S5E). (H) LV-transduced YFP+ MKs were cocultured with CSF1- or IL-33–induced YFPneg macrophages (MΦs) for 24 hours. (Graph) Quantification of TGF-β fluorescent reporter intensity in YFP+ MKs. Data are shown in box-and-whisker plots (midline, median; box, 25th and 75th percentiles; whiskers, 5th and 95th percentiles with outliers); ***P < 0.001. ns, not significant. Scale bars, 50 μm. (I) Quantification of the circularity of YFP+ MKs cocultured with CSF1- or IL-33–induced MFs. Data are shown in box-and-whisker plots, ***P < 0.001. (J and K) Quantification of YFP+ MKs invaded through Matrigel-coated membranes. (J) MKs were cultured with CSF1- or IL-33–induced MΦs or with (K) IL-33–induced MΦs transduced scramble control or Tgfb1 shRNA (n = 3). Data are shown as mean with SD, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 50 μm.

Journal: Science (New York, N.Y.)

Article Title: Tumor-initiating cells establish an IL-33-TGF-β niche signaling loop to promote cancer progression

doi: 10.1126/science.aay1813

Figure Lengend Snippet: (A) Histogram presentation of ST2 expression in FcεRIα+ and FcεRIαneg macrophages in SCC. (B) Flow cytometry analysis of FcεRIα and ST2 expression in CSF1- and IL-33–induced F4/80+ macrophages from bone marrow (BM)–derived cells (see fig. S5B). (C) qPCR analysis of CSF1-induced, IL-33–induced, and CSF1-induced→IL-4–treated macrophages in vitro (n = 3). Data are shown as relative mean expression with SD. (D) qPCR analysis of IL-33–induced macrophages treated with a MEK inhibitor (U0126, 0.5 –M), a JNK inhibitor (SP600125, 0.5 –M), a p38 inhibitor (SB202190, 0.5 –M), or an NF-κB inhibitor (BAY11-7082, 1 μM) during macrophage differentiation (n = 3). Data are shown as relative mean expression with SD and were analyzed with unpaired t test, **P = 0.0022. (E) qPCR analysis of IL-33–induced and IL-4–activated macrophages treated with the NF-κB inhibitor (n = 3). Data are shown as relative mean expression with SD and were analyzed with unpaired t test, ***P < 0.001. (F and G) qPCR analysis of IL-33–induced macrophages from ex vivo expanded hematopoietic progenitor cells that were LV transduced with scramble control, (F) Rela (NF-κB p65) shRNA, or (G) Fcer1a shRNA (see fig. S5E). (H) LV-transduced YFP+ MKs were cocultured with CSF1- or IL-33–induced YFPneg macrophages (MΦs) for 24 hours. (Graph) Quantification of TGF-β fluorescent reporter intensity in YFP+ MKs. Data are shown in box-and-whisker plots (midline, median; box, 25th and 75th percentiles; whiskers, 5th and 95th percentiles with outliers); ***P < 0.001. ns, not significant. Scale bars, 50 μm. (I) Quantification of the circularity of YFP+ MKs cocultured with CSF1- or IL-33–induced MFs. Data are shown in box-and-whisker plots, ***P < 0.001. (J and K) Quantification of YFP+ MKs invaded through Matrigel-coated membranes. (J) MKs were cultured with CSF1- or IL-33–induced MΦs or with (K) IL-33–induced MΦs transduced scramble control or Tgfb1 shRNA (n = 3). Data are shown as mean with SD, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 50 μm.

Article Snippet: To stain for ST2, biotinylated anti-ST2 antibody (1:1000, MD Biosciences, DJ8) was used.

Techniques: Expressing, Flow Cytometry, Derivative Assay, In Vitro, Ex Vivo, Transduction, shRNA, Whisker Assay, Cell Culture

(A) No difference is observed in the frequency of F4/80+ macrophages in live, CD45+ cells in control and Il33 KD tumors. Scramble control, n = 30; Il33 KD, n = 20. Data are shown as mean with SEM. ns, not significant. (B) F4/80+ macrophages in Il33 KD tumors have a smaller FcεRIα+ST2+ double-positive population than control tumors. (Graph) Quantification of FcεRIα+ST2+ macrophages. Scramble control, n = 27; Il33 KD, n = 20. Data are shown as mean with SEM and were analyzed with unpaired t test, ***P < 0.001. (C) Immunolabeling of tumor sections showing fewer FcεRIα+TGF-β+ cells in the stroma of Il33 KD tumors compared with control. (Graph) Quantification of FcεRIα+ cells in the stromal area within a 50-μm radius of tumor edges analyzed with unpaired t test, *P = 0.0394. (D) Immunolabeling of tumor sections showing that sST2-overexpressing (OE) tumors have fewer FcεRIα+ cells in the adjacent stroma. (Graph) Quantification of FcεRIα+ cells in the stromal area within a 50-μm radius of tumor edges analyzed with unpaired t test, ***P < 0.001. (E) HRASG12V-driven tumors overexpressing sST2 or control were sized. Control, n = 6; sST2 OE, n = 6. Approximation curves were drawn by applying the Michaelis-Menten kinetics curve. (F) Immunolabeling of K10 showing a sustained differentiation property in sST2-overexpressing tumors. (G) Immunolabeling of IL-33 and FcεRIα in Nrf2 (Nfe2l2) or Keap1 KD tumors. Whereas Nrf2 KD tumors show mostly nuclear IL-33, Keap1 KD tumors show cytoplasmic IL-33 localization. (Graph) Quantification of FcεRIα+ cells in the stromal area within a 50-μm radius of the tumor leading edges (n = 3-4). Data are shown in box-and-whisker plots, *P = 0.0394 (control versus Il33 KD) or 0.0372 (Nrf2 KD versus Keap1 KD). (H) Immunolabeling of tumor sections showing tdTomato+ immune cells infiltrated from the circulation at 7 d after injection. Note that control tdTomato+ cells expressed FcεRIα (arrowheads) but ST2 KD cells were largely negative (arrows). Magnified images are shown in fig. S6I. (Graph) Quantification of tdTomato+ cells in the stroma and FcεRIα-expressing tdTomato+ cells (n = 4) analyzed with unpaired t test, **P = 0.0019. (I) FcεRIα+ cell depletion by anti-FcεRIα antibody injection in vivo. Quantification of cells expressing ST2 (a surrogate marker for FcεRIα) within the stromal area in a 50-μm radius of tumor basal cells (n = 3), ***P < 0.001. (J) Quantification of TGF-β fluorescent reporter intensity in K5 basal cells. Intensity values in each image were normalized by the median values (set as 1) (n = 3) and analyzed with unpaired t test, ***P < 0.001. (K) Tumors were sized before and 1 week after anti-FcεRIα antibody injection. Isotype control, n = 3, 10 tumors; anti-FcεRIα, n = 3, 10 tumors. **P = 0.0016. (L) Model of TIC-driven feedforward mechanism of invasive SCC progression. TGF-β–responding TICs release IL-33 through the NRF2-mediated antioxidant response, which induces differentiation of immature myeloid cells into FcεRIα+ macrophages in their close proximity. In turn, FcεRIα+ macrophages send reciprocal paracrine TGF-β signaling to TICs to promote invasive progression and drug resistance of SCC, and further induce the release of IL-33, establishing a self-reinforcing niche signaling loop between TICs and FcεRIα+ macrophages. Dotted lines denote the tumor–stroma boundaries. Scale bar, 50 μm.

Journal: Science (New York, N.Y.)

Article Title: Tumor-initiating cells establish an IL-33-TGF-β niche signaling loop to promote cancer progression

doi: 10.1126/science.aay1813

Figure Lengend Snippet: (A) No difference is observed in the frequency of F4/80+ macrophages in live, CD45+ cells in control and Il33 KD tumors. Scramble control, n = 30; Il33 KD, n = 20. Data are shown as mean with SEM. ns, not significant. (B) F4/80+ macrophages in Il33 KD tumors have a smaller FcεRIα+ST2+ double-positive population than control tumors. (Graph) Quantification of FcεRIα+ST2+ macrophages. Scramble control, n = 27; Il33 KD, n = 20. Data are shown as mean with SEM and were analyzed with unpaired t test, ***P < 0.001. (C) Immunolabeling of tumor sections showing fewer FcεRIα+TGF-β+ cells in the stroma of Il33 KD tumors compared with control. (Graph) Quantification of FcεRIα+ cells in the stromal area within a 50-μm radius of tumor edges analyzed with unpaired t test, *P = 0.0394. (D) Immunolabeling of tumor sections showing that sST2-overexpressing (OE) tumors have fewer FcεRIα+ cells in the adjacent stroma. (Graph) Quantification of FcεRIα+ cells in the stromal area within a 50-μm radius of tumor edges analyzed with unpaired t test, ***P < 0.001. (E) HRASG12V-driven tumors overexpressing sST2 or control were sized. Control, n = 6; sST2 OE, n = 6. Approximation curves were drawn by applying the Michaelis-Menten kinetics curve. (F) Immunolabeling of K10 showing a sustained differentiation property in sST2-overexpressing tumors. (G) Immunolabeling of IL-33 and FcεRIα in Nrf2 (Nfe2l2) or Keap1 KD tumors. Whereas Nrf2 KD tumors show mostly nuclear IL-33, Keap1 KD tumors show cytoplasmic IL-33 localization. (Graph) Quantification of FcεRIα+ cells in the stromal area within a 50-μm radius of the tumor leading edges (n = 3-4). Data are shown in box-and-whisker plots, *P = 0.0394 (control versus Il33 KD) or 0.0372 (Nrf2 KD versus Keap1 KD). (H) Immunolabeling of tumor sections showing tdTomato+ immune cells infiltrated from the circulation at 7 d after injection. Note that control tdTomato+ cells expressed FcεRIα (arrowheads) but ST2 KD cells were largely negative (arrows). Magnified images are shown in fig. S6I. (Graph) Quantification of tdTomato+ cells in the stroma and FcεRIα-expressing tdTomato+ cells (n = 4) analyzed with unpaired t test, **P = 0.0019. (I) FcεRIα+ cell depletion by anti-FcεRIα antibody injection in vivo. Quantification of cells expressing ST2 (a surrogate marker for FcεRIα) within the stromal area in a 50-μm radius of tumor basal cells (n = 3), ***P < 0.001. (J) Quantification of TGF-β fluorescent reporter intensity in K5 basal cells. Intensity values in each image were normalized by the median values (set as 1) (n = 3) and analyzed with unpaired t test, ***P < 0.001. (K) Tumors were sized before and 1 week after anti-FcεRIα antibody injection. Isotype control, n = 3, 10 tumors; anti-FcεRIα, n = 3, 10 tumors. **P = 0.0016. (L) Model of TIC-driven feedforward mechanism of invasive SCC progression. TGF-β–responding TICs release IL-33 through the NRF2-mediated antioxidant response, which induces differentiation of immature myeloid cells into FcεRIα+ macrophages in their close proximity. In turn, FcεRIα+ macrophages send reciprocal paracrine TGF-β signaling to TICs to promote invasive progression and drug resistance of SCC, and further induce the release of IL-33, establishing a self-reinforcing niche signaling loop between TICs and FcεRIα+ macrophages. Dotted lines denote the tumor–stroma boundaries. Scale bar, 50 μm.

Article Snippet: To stain for ST2, biotinylated anti-ST2 antibody (1:1000, MD Biosciences, DJ8) was used.

Techniques: Immunolabeling, Whisker Assay, Injection, Expressing, In Vivo, Marker

Journal: Immunity

Article Title: Interleukin-33 induces the enzyme tryptophan hydroxylase 1 to promote inflammatory group 2 innate lymphoid cell-mediated immunity

doi: 10.1016/j.immuni.2020.02.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse ST2 (RMST2-2)-Biotinylated , eBioscience , Cat#13-9333-82.

Techniques: Control, Modification, Virus, Recombinant, Lysis, Electron Microscopy, Staining, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software, Real-time Polymerase Chain Reaction, Spectrophotometry

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