Journal: Frontiers in Pharmacology

Article Title: Ruxolitinib alleviates DSS-induced acute ulcerative colitis by inhibiting STAT1 phosphorylation and reducing MDSC infiltration

doi: 10.3389/fphar.2025.1572534

Figure Lengend Snippet: Effect of ruxolitinib on JAK/STAT signaling pathway-related genes and inhibition of interferon (IFN)-γ-Induced STAT1 activation by ruxolitinib. (A) Heatmap of transcription levels (fold-changes) of the JAK/STAT signaling-related genes in CCD841 and Jurkat cells after treatment with IFN-γ and ruxolitinib. (B) Nuclear extracts prepared from CCD841 cells with or without IFN-γ activation and analyzed for STAT1 activation using electrophoretic mobility shift assay (EMSA) with the STAT1-binding consensus sequence DNA probe. Ruxolitinib at 50, 250, and 500 nM was incubated with STAT1-DNA complexes. RUX: ruxolitinib.

Article Snippet: DSS was purchased from MP Biomedicals; Ruxolitinib was purchased from Selleck; The following antibodies were used: APC-conjugated anti-CD11b and PE-conjugated anti-Gr-1 (BioLegend), STAT1 and phosphorylated STAT1 (Tyr701) (Proteintech), phosphorylated STAT1 (Ser727) (Cell Signaling Technology).

Techniques: Inhibition, Activation Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Sequencing, Incubation

Journal: Frontiers in Pharmacology

Article Title: Ruxolitinib alleviates DSS-induced acute ulcerative colitis by inhibiting STAT1 phosphorylation and reducing MDSC infiltration

doi: 10.3389/fphar.2025.1572534

Figure Lengend Snippet: Inhibition of STAT1 phosphorylation by ruxolitinib in immune cells and normal intestinal epithelial cells. (A) Western blot analysis of p-STAT1Y701, p-STAT1S727, STAT1, and β-actin in Jurkat, RAW and CCD841 cells treated with 50 IU/mL IFN-γ for the indicated times. (B–D) Quantitative analysis of p-STAT1Y701, p-STAT1S727, STAT1 levels in (B) Jurkat, (C) RAW, and (D) CCD841 cells from (A) . (E) Western blot analysis of p-STAT1Y701, p-STAT1S727, STAT1, and β-actin in cells pretreated with IFN-γ (8 h) followed by Ruxolitinib (RUX, 24 h) at indicated concentrations (μM). (F–H) Quantitative analysis of p-STAT1Y701, p-STAT1S727, STAT1 levels in (F) Jurkat, (G) RAW, and (H) CCD841 cells from (E) . # P < 0.05, ## P < 0.01, ### P < 0.001 vs. 0 μM RUX (IFN-γ-treated). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, * P < 0.001 vs. the group with IFN-γ concentration at 0; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. the IFN-γ-treated group with RUX concentration at 0. RUX: ruxolitinib.

Article Snippet: DSS was purchased from MP Biomedicals; Ruxolitinib was purchased from Selleck; The following antibodies were used: APC-conjugated anti-CD11b and PE-conjugated anti-Gr-1 (BioLegend), STAT1 and phosphorylated STAT1 (Tyr701) (Proteintech), phosphorylated STAT1 (Ser727) (Cell Signaling Technology).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Concentration Assay

Journal: iScience

Article Title: AF6 regulates intestinal IgA via crosstalk between intestinal epithelial cells and immune cells in inflammatory bowel disease

doi: 10.1016/j.isci.2025.112658

Figure Lengend Snippet: AF6 regulates the expression of MHC II by modulating the expression of STAT1 in intestinal epithelial cells (IECs) (A) The protein levels of components of the IFN-γ-related signaling pathway were detected by immunoblotting of colon tissues from Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. (B) qPCR was used to assess STAT1 transcript levels in colon tissues from Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. (C and D) (C) The mRNA levels of STAT1 and its downstream target genes (as assessed by qPCR) and (D) the protein levels of STAT1 and proteins encoded by its downstream target genes (as assessed by immunoblotting) were determined in organoids generated from colon tissues of Af6 f/f and Af6 ΔIEC mice, as measured before and after IFN-γ treatment. (E) Co-immunoprecipitation (co-IP) was used to detect endogenous interactions between AF6 and IRF1 in IECs. (F) 293T cells were co-transfected with constructs encoding hemagglutinin-tagged AF6 (HA-AF6) and Flag peptide-tagged IRF1 (Flag-IRF1); their interaction domains were detected by co-IP. (G) Immunoblotting was used to assess the expression of IRF1 and proteins encoded by its downstream genes in HT29 cells with or without IFN-γ exposure. (H) HT29 cells were transfected with constructs encoding AF6 with no or 3 nuclear localization signaling (NLS) domains (ΔNLS-AF6 and 3×NLS-AF6, respectively), and the expression and localization of IRF1 and proteins encoded by its downstream genes were assessed by immunoblotting following the separation of the nuclear and cytoplasmic fractions. Data are expressed as mean ± SEM. Pairwise comparisons between groups were conducted using two-tailed non-paired Student’s t tests. p -values were shown in the panel, and p < 0.05 indicates a significant difference.

Article Snippet: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb , Cell Signaling Technology , Cat#9167.

Techniques: Expressing, Western Blot, Generated, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Construct, Two Tailed Test