Journal: Frontiers in Immunology

Article Title: ATF3 Sustains IL-22-Induced STAT3 Phosphorylation to Maintain Mucosal Immunity Through Inhibiting Phosphatases

doi: 10.3389/fimmu.2018.02522

Figure Lengend Snippet: List of the materials used in the study.

Article Snippet: Cells were thoroughly washed with PBS before staining with a blocking antibody (CD16/32) followed by intracellular staining with PE-pSTAT3 and PE-IgG2b isotype control antibodies (eBioscience).

Techniques: Concentration Assay, Purification, Flow Cytometry, Control, Immunofluorescence, Western Blot, Membrane, Recombinant, Gentle, Staining, In Situ, TUNEL Assay, cDNA Synthesis, Real-time Polymerase Chain Reaction, Bradford Protein Assay, DNA Labeling, Cell Stimulation, Isolation, Modification, Cell Culture, In Vitro, Transfection, CRISPR, In Vivo, RNA Extraction, SYBR Green Assay, Sequencing

Journal: Frontiers in Immunology

Article Title: ATF3 Sustains IL-22-Induced STAT3 Phosphorylation to Maintain Mucosal Immunity Through Inhibiting Phosphatases

doi: 10.3389/fimmu.2018.02522

Figure Lengend Snippet: ATF3 promotes IL-22-induced STAT3 phosphorylation by suppressing phosphatases. (A) Freshly isolated ileum crypts, or (B) ileum organoids at day 6 of culture, were stimulated with IL-22, followed by fixation and intracellular staining of phospho-STAT3, and analyzed by flow cytometry. Western blot analysis of (C) IL-22-stimulated CMT93 cells, or (D) IL-22-stimuated colon fragments isolated from the indicated mice, for the expression of the indicated proteins. (E) Quantitative real-time PCR analysis of IL-22R1 and IL-10R2 mRNA levels in freshly isolated ileum crypts from mice. (F) Flow cytometry analysis of IL-22R1 in freshly isolated ileum crypt cells gated on the CD45 − EpCAM + population. (G,H) Western blot analysis of unstimulated or IL-22-stimulated CMT93 cells for the indicated proteins. ATF3 −/− CMT93 cells with SHP2 knockdown (ATF3 −/− SHP2 KD ) were indicated. Images were representative of four independent experiments (G–H) . Results were from two independent experiments (A–F) . “n” refers to the number of mice analyzed (A,B,E,F) . Statistical analysis was done by multiple comparison in Two-way ANOVA test using Prism software. * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Cells were thoroughly washed with PBS before staining with a blocking antibody (CD16/32) followed by intracellular staining with PE-pSTAT3 and PE-IgG2b isotype control antibodies (eBioscience).

Techniques: Phospho-proteomics, Isolation, Staining, Flow Cytometry, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Knockdown, Comparison, Software

Journal: Frontiers in Immunology

Article Title: ATF3 Sustains IL-22-Induced STAT3 Phosphorylation to Maintain Mucosal Immunity Through Inhibiting Phosphatases

doi: 10.3389/fimmu.2018.02522

Figure Lengend Snippet: ATF3 regulates IL-6-pSTAT3 signaling in intestinal Th17 cells. Flow cytometry analysis of phospho-STAT3 in (A) IL-6 or IL-22 stimulated freshly isolated ileum crypts or IL-6-stimulated peripheral blood mononuclear cell (PBMC) from wild-type mice, or in (B) IL-6-stimulated PBMC from wild-type and ATF3-deficient mice. (C) Flow cytometry analysis of intracellular IL-17A and IL-22 expression in naïve lamina propria T cells from the indicated mice. Cells were treated with PMA, ionomycin and IL-23 in the presence of BFA for 4 h before analysis and gated on live CD45 + EpCAM − Lin − CD3 + population as shown. (D) Quantitative real-time PCR analysis of IL-17A and IL-22 mRNA levels in freshly isolated lamina propria (LPL) cells, mesenteric lymph nodes (mLN), or splenocytes. (E) Model of ATF3-mediated mucosal immunity via cross-regulation between IL-22-pSTAT3 signaling in epithelium (associated with AMP production and epithelial fucosylation) and IL-6-pSTAT3 signaling in Th17 cells (associated with signature IL-17A and IL-22 production). “n” refers to the number of mice analyzed. Statistical analysis was done by multiple comparison in Two-way ANOVA test using Prism software. * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Cells were thoroughly washed with PBS before staining with a blocking antibody (CD16/32) followed by intracellular staining with PE-pSTAT3 and PE-IgG2b isotype control antibodies (eBioscience).

Techniques: Flow Cytometry, Isolation, Expressing, Real-time Polymerase Chain Reaction, Comparison, Software

Journal: Frontiers in Immunology

Article Title: Prolactin promotes proliferation of germinal center B cells, formation of plasma cells, and elevated levels of IgG3 anti-dsDNA autoantibodies

doi: 10.3389/fimmu.2022.1017115

Figure Lengend Snippet: PRL signals through the long receptor isoform and STAT1 in B-GCs. B cells purified from female MRL/lpr mice were differentiated into B-GCs for 48 h ( in vitro ), and B-GC cells were obtained directly from 15-weeks-old female MRL/lpr mice ( in vivo ). (A) The relative expression and identity of the PRL receptor isoforms were determined by real-time (RT)-PCR (normalized to the endogenous gene β-actin and using the breast cancer cell line 4T1 as a control for the expression of both isoforms). To determine the percentage of phosphorylated cells, B-GCs were differentiated for 48 h, left in medium for 8 h and then stimulated for 30 min with PRL to determine (B) zebra plots of pSTAT1, (C) the percentage of pSTAT1, (D) MFI of pSTAT1 (E) the percentage of pAKT, (F) MFI of pAKT, and the percentages of (G) pSTAT3, (H) pSTAT5, and (I) pERK. Six independent experiments were performed. Pooled data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ns, no statistical differences using a Student’s T test for paired samples.

Article Snippet: The cells were fixed with 1x BD Phosflow Lyse/Fix Buffer 5x (BD Biosciences)/10 minutes, with BD Phosflow Perm Buffer III (BD Biosciences); the cells were permeabilized to determine intracellular STAT1 (anti-pSTAT1 PE, clone A15158B, BioLegend), STAT3 (anti-pSTAT3 PE, clone 13A3-1, BioLegend), and STAT5 (anti-pSTAT5 PE, clone SRBCZX, eBioscience) phosphorylation.

Techniques: Purification, In Vitro, In Vivo, Expressing, Quantitative RT-PCR

Journal: Acta Pharmacologica Sinica

Article Title: Ursolic acid ameliorates autoimmune arthritis via suppression of Th17 and B cell differentiation

doi: 10.1038/aps.2014.58

Figure Lengend Snippet: Primer list used for this experiment.

Article Snippet: To identify Th17 cells, tissue sections were stained with anti-IL-17A-FITC (eBioscience), -CD4-APC (eBioscience), and -pSTAT3-PE (Tyr705 or Ser727; BD) antibodies overnight at 4 °C, and analysed using an LSM 510 Meta confocal microscopy system (Carl Zeiss, Germany).

Techniques: Sequencing

Journal: Acta Pharmacologica Sinica

Article Title: Ursolic acid ameliorates autoimmune arthritis via suppression of Th17 and B cell differentiation

doi: 10.1038/aps.2014.58

Figure Lengend Snippet: UA decreases the expression of proinflammatory cytokines and oxidative stress-related genes in the joints of CIA mice. Ankle joints (6 per group) of CIA mice treated with either UA or vehicle control were immunostained for (A) IL-1β, IL-6, TNF-α, (B) IL-17, IL-21, nitrotyrosine, and iNOS, or (C) isotype control. Representative data are shown.

Article Snippet: To identify Th17 cells, tissue sections were stained with anti-IL-17A-FITC (eBioscience), -CD4-APC (eBioscience), and -pSTAT3-PE (Tyr705 or Ser727; BD) antibodies overnight at 4 °C, and analysed using an LSM 510 Meta confocal microscopy system (Carl Zeiss, Germany).

Techniques: Expressing

Journal: Acta Pharmacologica Sinica

Article Title: Ursolic acid ameliorates autoimmune arthritis via suppression of Th17 and B cell differentiation

doi: 10.1038/aps.2014.58

Figure Lengend Snippet: UA reduces STAT3 phosphorylation and decreases the frequency of Th17 cells within the population of CD4+ T cells in CIA mice. (A) Spleens of UA- or vehicle-treated CIA mice were subjected to immunostaining for CD4+IL-17+, CD4+CD25+, and CD4+foxp3+. (B) The frequency CD4+IL-17+ cells among ex vivo total splenocytes was assessed using flow cytometry. (C) The mRNA expression of IL-17, IL-21, and RORγt was determined by real-time RT-PCR in cells obtained from the spleen (Sp) or draining lymph nodes (LN). (D) Spleens of UA- or vehicle-treated CIA mice were subjected to immunostaining of CD4+pSTAT3 Y705+ or CD4+pSTAT3 S727+ cells. The data are presented as the mean±SD of 3 animals per group. bP<0.05, cP<0.01.

Article Snippet: To identify Th17 cells, tissue sections were stained with anti-IL-17A-FITC (eBioscience), -CD4-APC (eBioscience), and -pSTAT3-PE (Tyr705 or Ser727; BD) antibodies overnight at 4 °C, and analysed using an LSM 510 Meta confocal microscopy system (Carl Zeiss, Germany).

Techniques: Immunostaining, Ex Vivo, Flow Cytometry, Expressing, Quantitative RT-PCR

Journal: Acta Pharmacologica Sinica

Article Title: Ursolic acid ameliorates autoimmune arthritis via suppression of Th17 and B cell differentiation

doi: 10.1038/aps.2014.58

Figure Lengend Snippet: UA inhibits Th17 differentiation in vitro. CD4+ T cells were isolated from the spleens of DBA/1J mice and incubated for 3 d under Th17-polarizing conditions (0.5 μg/mL anti-CD3 mAb, 1 μg/mL anti-CD28 mAb, 2 μg/mL anti-IL-4 Ab, 2 μg/mL anti-IFN-γ Ab, 20 ng/mL IL-6, and 2 ng/mL TGF-β) in the presence or absence of UA. (A) UA doses ≤10 μmol/L did not affect cell viability. (B) The frequency of CD4+IL-17+ T cells was measured by flow cytometry. (C) The expression of IL-17, IL-21, RORγt, and Foxp3 was analysed by real-time PCR. The data are presented as the mean±SEM of three independent experiments. bP<0.05, cP<0.01 relative to the control mice.

Article Snippet: To identify Th17 cells, tissue sections were stained with anti-IL-17A-FITC (eBioscience), -CD4-APC (eBioscience), and -pSTAT3-PE (Tyr705 or Ser727; BD) antibodies overnight at 4 °C, and analysed using an LSM 510 Meta confocal microscopy system (Carl Zeiss, Germany).

Techniques: In Vitro, Isolation, Incubation, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction