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