Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: STAT6-dependent regulation of Th9 development

doi: 10.4049/jimmunol.1102840

Figure Lengend Snippet: A, Naïve CD4+T cells from WT mice were activated with anti-CD3 and anti-CD28 and cultured with IL-4 and anti-IFN-γ (Th2 conditions) or with IL-4, TGF-β and anti-IFN-γ (Th9 conditions). Each day during differentiation, cells were stained for intracellular-phospho-STAT3 and phospho-STAT6. The bar graphs represent percentages of cells (top) and mean fluorescent intensity (MFI, bottom). Data are average ± S.E.M of 6 mice from 3 experiments.B, C. Naïve CD4+T cells from WT and Stat6−/− (top), WT and Stat3CD4−/− (middle), and WT and Parp14−/− mice (bottom) were cultured under Th9 conditions. After 5d cells were harvested and stimulated with PMA and Ionomycin before intracellular staining for cytokines IL-9 and IL-4 was performed (B) or stimulated with anti-CD3 and 24h later cell-free supernatant was collected and IL-9 production was assessed by ELISA (C). Data are representative of 3 experiments with similar results.*, p<0.05. ***, p<0.001.

Article Snippet: Cell free supernatant was collected and the amount of IL-9 was assessed by ELISA using anti-IL-9 capture antibody (D8402E8; BD Biosciences) and biotin-labeled anti-IL-9 (D9302C12; Biolegend) as the secondary antibody.

Techniques: Cell Culture, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: STAT6-dependent regulation of Th9 development

doi: 10.4049/jimmunol.1102840

Figure Lengend Snippet: A-C. WT and Il4−/− naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and cultured under Th9 cell conditions for 48h before being transduced with control or GATA3-expressing retroviruses (A), control or c-Maf expressing retroviruses (B), or control or JunB-expressing retroviruses (C). After 5d in culture cells were stimulated with PMA and Ionomycin for intracellular staining of IL-9 and IL-4. Data are representative of 2-3 independent experiments with similar results.*, p<0.05. **, p<0.01.

Article Snippet: Cell free supernatant was collected and the amount of IL-9 was assessed by ELISA using anti-IL-9 capture antibody (D8402E8; BD Biosciences) and biotin-labeled anti-IL-9 (D9302C12; Biolegend) as the secondary antibody.

Techniques: Cell Culture, Transduction, Expressing, Staining

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: STAT6-dependent regulation of Th9 development

doi: 10.4049/jimmunol.1102840

Figure Lengend Snippet: A, Naïve CD4+T cells from WT mice were analyzed directly ex vivo (naïve) or cultured under Th1, Th2, Th17, Th9, and Treg cell conditions for 5d. After RNA isolation, quantitative PCR was performed for the indicated genes. Data are representative of 2 experiments with similar results. B, Total cell lysates were prepared from Th1, Th2, and Th9 cells and immunoblot was performed for Runx3, β-actin was used as loading control. C, WT naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and cultured under Th9 cell conditions for 48h before being transduced with control or Runx3-expressing retroviruses and control or T-bet expressing retroviruses. Cells were harvested after 5d in culture and stimulated with PMA and ionomycin before intracellular IL-9 and IL-4 staining. Data are average ± S.D of 2 experiments with 4 mice. D, Naïve CD4+T cells from WT and Tbx21−/− (bottom left) or WT and Runx3fl/fl-CD4-Cre (bottom right) were cultured in Th9 cell conditions. After 5d cells were harvested and stimulated with PMA and Ionomycin before intracellular IL-9 and IL-4 staining. Data are representative of 2-3 experiments with similar results. E, WT and Stat6−/−naïve CD4+T cells were cultured under Th9 cell conditions for 5d. Cells were harvested and quantitative PCR was performed for Runx3and Tbx21.*, p<0.05. **, p<0.01.

Article Snippet: Cell free supernatant was collected and the amount of IL-9 was assessed by ELISA using anti-IL-9 capture antibody (D8402E8; BD Biosciences) and biotin-labeled anti-IL-9 (D9302C12; Biolegend) as the secondary antibody.

Techniques: Ex Vivo, Cell Culture, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Transduction, Expressing, Staining

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: STAT6-dependent regulation of Th9 development

doi: 10.4049/jimmunol.1102840

Figure Lengend Snippet: A, WT naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and were cultured with increasing doses of TGF-β in the presence (Th9) or absence (Treg) of IL-4 for 5d. Cells were then stained for intracellular Foxp3 (top) and IL-9 (middle). Differentiated Th9 cells were stimulated with anti-CD3 and 24h later IL-9 production was assessed by ELISA (bottom). B, Naïve CD4+T cells were cultured under Th9 and Treg cell conditions as in A (left) or cultured with 2 ng/ml TGF-β and increasing doses of IL-4 (Th9, right) for 5d. After differentiation, RNA was isolated and Sfpi1 expression was measured by quantitative PCR. Data are representative of 2 experiments with similar results. C, WT and Stat6−/− naïve CD4+T cells were cultured under Th9 cell conditions for 5d and Foxp3 expression was measured using qPCR. Data are average ± S.D of 4 mice from 2 experiments. D, Naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and cultured in Th9 cell conditions for 48h before being transduced with control or Foxp3-expressing retroviruses. After 5d in culture cells were stimulated with PMA and Ionomycin and intracellularly stained for IL-9 and IL-17A. Data are representative of 2 experiments with similar results.E, Schematic showing a summary of a transcription factor network in Th9 cells.*, p<0.05.

Article Snippet: Cell free supernatant was collected and the amount of IL-9 was assessed by ELISA using anti-IL-9 capture antibody (D8402E8; BD Biosciences) and biotin-labeled anti-IL-9 (D9302C12; Biolegend) as the secondary antibody.

Techniques: Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Real-time Polymerase Chain Reaction, Transduction

Journal: iScience

Article Title: PTEN acts as a crucial inflammatory checkpoint controlling TLR9/IL-6 axis in B cells

doi: 10.1016/j.isci.2024.110388

Figure Lengend Snippet: Loss of PTEN in B2 B cells promotes TLR9-mediated IL-6 production (A) Overlaid curves showing the expression levels of IL-6, IL-9, IL-17, CCL5, G-CSF, IL-4, IL-10, IFN-γ, and TNF-α in splenic B cells from the indicated mice ( n = 3 male mice/group) at 25–33 weeks was determined by flow cytometry. Cells were left unstimulated or stimulated with CpG, LPS, P+I, CpG+P+I, or LPS+P+I, in vitro for 24 h. (B) FACS profiles of IL-6 versus CD19 in splenic B cells from the indicated mice ( n = 9 male mice/group) at 10 w. (C) Percentages of IL-6 + B cells in splenic B cells as described in (B). (D) Histograms showing IL-6 produced by FO (left) and MZ (right) B cells, which were left unstimulated (resting) or stimulated with CpG, CL307, LPS, and poly(I:C) in vitro for 24 h, as determined by ELISA. Each group containing 3 male mice. (E) Histograms showing IL-6 produced by FO (left), MZ (middle), and B1a (right) B cells from the indicated mice that were left untreated (resting) or stimulated with CpG in vitro for 24 h, as determined by ELISA. Each group containing 4 male mice. (F) Histograms showing IL-6 produced by FO (left) and MZ (right) B cells, which were left untreated (resting) or stimulated with CpG in the absence or presence of inhibitors (inh.) targeting NF-κB p65 or p50, respectively, for 24 h, as determined by ELISA. Each group containing 4 male mice. (G) Kaplan-Meier curves of female CD23-control mice (black line, n = 16), female CD23-cKO mice (red line, n = 24), and female CD23-cKO/IL-6 −/− double knockout mice (blue line, n = 19). The samples were compared using an unpaired two-tailed t test; ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.0005, and the data are presented as mean ± SEM.

Article Snippet: Alexa Fluor® 647 Armenian Hamster anti-Mouse IL-9 (clone D9302C12) , BD Biosciences , Cat#561464; RRID: AB_10644410.

Techniques: Expressing, Flow Cytometry, In Vitro, Produced, Enzyme-linked Immunosorbent Assay, Control, Double Knockout, Two Tailed Test

Journal: iScience

Article Title: PTEN acts as a crucial inflammatory checkpoint controlling TLR9/IL-6 axis in B cells

doi: 10.1016/j.isci.2024.110388

Figure Lengend Snippet:

Article Snippet: Alexa Fluor® 647 Armenian Hamster anti-Mouse IL-9 (clone D9302C12) , BD Biosciences , Cat#561464; RRID: AB_10644410.

Techniques: Recombinant, Western Blot, Staining, In Situ, Antibody Labeling, Enzyme-linked Immunosorbent Assay, Software

Journal: OncoTargets and therapy

Article Title: Effect of Th9/IL-9 on the growth of gastric cancer in nude mice

doi: 10.2147/OTT.S197816

Figure Lengend Snippet: Effect of rIL-9 on the factors in serum of SGC-7901-xenografted nude mices. rIL-9 could inhibit the levels of IL-4 ( A ), IL-9 ( B ), IL-10 ( C ), IL-25 ( D ), VEGF ( E ), and TGF-β ( F ) in serum of SGC-7901-xenografted nude mice. Notes: Data are shown as mean±SD. * P <0.05 vs Control group, # P <0.05 vs Model group.

Article Snippet: They were then stained with PE anti-mouse IL-9 or PE anti-mouse IL-17 antibody (BD Bioscience PharMingen).

Techniques:

Journal: OncoTargets and therapy

Article Title: Effect of Th9/IL-9 on the growth of gastric cancer in nude mice

doi: 10.2147/OTT.S197816

Figure Lengend Snippet: Effect of rIL-9 on the migration and invasion of SGC-7901 cell ( A ). IL-9 can promote tumor migration ( B ) and invasion ( C ) (400×). Data were shown as mean±SD. # P <0.05 vs model group.

Article Snippet: They were then stained with PE anti-mouse IL-9 or PE anti-mouse IL-17 antibody (BD Bioscience PharMingen).

Techniques: Migration

Journal: OncoTargets and therapy

Article Title: Effect of Th9/IL-9 on the growth of gastric cancer in nude mice

doi: 10.2147/OTT.S197816

Figure Lengend Snippet: Effect of rIL-9 on the expression of IL-9 and CD34 proteins in gastric cancer tissue ( A ). rIL-9 could inhibit the expression of IL-9 ( B ) and CD34 ( C ). Notes: Data are shown as mean±SD. # P <0.05 vs Model group.

Article Snippet: They were then stained with PE anti-mouse IL-9 or PE anti-mouse IL-17 antibody (BD Bioscience PharMingen).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: Effect of Th9/IL-9 on the growth of gastric cancer in nude mice

doi: 10.2147/OTT.S197816

Figure Lengend Snippet: Effect of rIL-9 on the mRNA expression of IL-9, IL-21, and PU.1 in gastric cancer tissue. rIL-9 can inhibit the mRNA expression of IL-9, IL-21, and PU.1. Notes: Data are shown as mean±SD. # P <0.05 vs Model group.

Article Snippet: They were then stained with PE anti-mouse IL-9 or PE anti-mouse IL-17 antibody (BD Bioscience PharMingen).

Techniques: Expressing

Journal: Veterinary Research

Article Title: Identification of excretory and secretory proteins from Haemonchus contortus inducing a Th9 immune response in goats

doi: 10.1186/s13567-022-01055-8

Figure Lengend Snippet: Th9-cell sorting by flow cytometry . A Th9 cells induced by HcESPs in vitro. PBMCs were treated with PBS (0 μg/mL HcESPs as the control) ( A Panel 1) or 80 μg/mL HcESPs ( A Panel 2). Th9 cells were sorted by flow cytometry using CD2 + CD4 + IL-9 + IL-10 + as the gate. B – E Percentages of Th9 cells at 7 ( B ), 15 ( C ), 35 ( D ) and 50 ( E ) dpi. Samples were collected from the blank group ( B–E Panel 1) and challenge group ( B–E Panel 2) at 7, 15, 35 and 50 dpi.

Article Snippet: Finally, the cells were permeabilized by adding BD Perm/Wash buffer (BD Biosciences, Becton, USA) and stained with PE-Cy5-labelled anti-IL-9 (GenScript, New Jersey, USA) and PE-labelled anti-IL-10 antibodies (BD, Pharmingen, USA) for 30 min.

Techniques: FACS, Flow Cytometry, In Vitro

Journal: Veterinary Research

Article Title: Identification of excretory and secretory proteins from Haemonchus contortus inducing a Th9 immune response in goats

doi: 10.1186/s13567-022-01055-8

Figure Lengend Snippet: Effects of rHcDR on Th9 cells and IL-9 expression in vitro. A Agarose gel electrophoresis of the HcDR gene. Lane 1: amplification products of the HcDR gene. Lane M: DNA molecular weight marker. B Purification of rHcDR. Lane 2: before purification. Lane 3: purified rHcDR. Lane M: standard protein molecular marker. C Western blot. Lane 4: rHcDR recognized by serum from an H. contortus -infected goat. Lane 5: No recognition by normal serum. Lane M: standard protein molecular marker. D The effects of rHcDR on Th9-cell proliferation. Goat PBMCs were treated with different concentrations of rHcDR (0, 5, 10, 20, 40 and 60 μg/mL). Th9 cells were detected by flow cytometry using staining with antibodies specific for typical intracellular cytokines (IL-9 and IL-10). E Proportions of Th9 cells observed with different concentrations of rHcDR (0, 5, 10, 20, 40 and 60 μg/mL). Data are presented as the mean ± SD and are representative of triplicate experiments ( *p < 0.05, ****p < 0.0001). F Fold change in relative IL-9 mRNA expression. Goat PBMCs were stimulated with different concentrations of rHcDR. The significance level was set at *p < 0.05, **p < 0.01, or ****p < 0.0001, and “ns” indicates non-significance compared with the control (blank). Data are representative of three independent experiments.

Article Snippet: Finally, the cells were permeabilized by adding BD Perm/Wash buffer (BD Biosciences, Becton, USA) and stained with PE-Cy5-labelled anti-IL-9 (GenScript, New Jersey, USA) and PE-labelled anti-IL-10 antibodies (BD, Pharmingen, USA) for 30 min.

Techniques: Expressing, In Vitro, Agarose Gel Electrophoresis, Amplification, Molecular Weight, Marker, Purification, Western Blot, Infection, Flow Cytometry, Staining

Journal: Veterinary Research

Article Title: Identification of excretory and secretory proteins from Haemonchus contortus inducing a Th9 immune response in goats

doi: 10.1186/s13567-022-01055-8

Figure Lengend Snippet: Effects of rHcGATA on Th9-cell proliferation and IL-9 transcription. A Agarose gel electrophoresis of the HcGATA gene. Lane 1: reverse transcription PCR products of HcGATA; Lane M: DNA molecular weight marker. B Purification of rHcGATA. Lane 2: rHcGATA before purification. Lane 3: purified rHcGATA. Lane M: standard protein molecular marker. C Western blot. Lane 4: rHcGATA protein recognized by serum from an H. contortus -infected goat. Lane 5: rHcGATA was not recognized by normal serum. Lane M: standard protein molecular marker. D The effects of HcGATA on the proliferation of Th9 cells in vitro. PBMC-derived Th9 cells treated with a control (0 μg/mL) or different concentrations of HcGATA (5, 10, 20, 40, 60 μg/mL) were tested by flow cytometry using antibodies specific for typical intracellular cytokines (IL-9 and IL-10). E Proportions of Th9 cells observed with different concentrations of HcGATA (0, 5, 10, 20, 40 and 60 μg/mL). Data are presented as the mean ± SD representative of triplicate experiments (ns p > 0.05, *p < 0.05, ****p < 0.0001). F Fold change in relative IL-9 mRNA expression. Goat PBMCs were stimulated with different concentrations of rHcGATA. The significance level was set at ***p < 0.001, or ****p < 0.0001, and “ns” indicates non-significance compared with the control group. Data are representative of three independent experiments.

Article Snippet: Finally, the cells were permeabilized by adding BD Perm/Wash buffer (BD Biosciences, Becton, USA) and stained with PE-Cy5-labelled anti-IL-9 (GenScript, New Jersey, USA) and PE-labelled anti-IL-10 antibodies (BD, Pharmingen, USA) for 30 min.

Techniques: Agarose Gel Electrophoresis, Molecular Weight, Marker, Purification, Western Blot, Infection, In Vitro, Derivative Assay, Flow Cytometry, Expressing