Journal: Frontiers in Immunology

Article Title: Transcriptional inhibition of STAT1 functions in the nucleus alleviates Th1 and Th17 cell-mediated inflammatory diseases

doi: 10.3389/fimmu.2022.1054472

Figure Lengend Snippet: Generation of nucleus-deliverable ndSTAT1-TMD and verification of its intra-nuclear transduction kinetics without cellular cytotoxicity. (A) Protein structure of STAT1-TMD without Hph-1-PTD, ndSTAT-TMD, and ndSTAT1-TMD (V426D, T427D) with mutated valine 426, and threonine 427. PTD, protein transduction domain; nd, nucleus-transducible; TMD, transcription modulation domain; DBD, DNA binding domain; ND, N-terminal domain; LD, linker domain; SH2, SCR2 homology domain; TAD, transactivation domain. (B) The identity of the purified STAT1-TMD, ndSTAT1-TMD, and ndSTAT1-TMD (V426D, T427D) was confirmed by western blot (left panel) or SDS-PAGE (right panel). (C, D) Dose-dependent and time-dependent intra-nuclear delivery of ndSTAT1-TMD (0.5-2 μM) or STAT1-TMD (2 μM) into mouse splenocytes. (E) Intracellular stability of ndSTAT1-TMD after transduction into mouse splenocytes. (F) Intracellular localization of ndSTAT1-TMD in cultured mouse splenocytes. Representative immunofluorescence 63X (left panel) and 189X (right panel) images using a confocal microscope. Scale bar = 10 μm. (G) Evaluation of cellular cytotoxicity of ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) in mouse splenocytes analyzed by CCK-8 assay. (H, I) The level of the induced expression of CD25, CD69, or IL-2 secretion in CD4 + T cells activated by anti-CD3ϵ antibody and anti-CD28 antibody in the presence of ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) was analyzed by flow cytometry and ELISA. The graphs are represented as mean ± SEM (n=3), and the statistical analysis was examined using Student’s t-test. ns, not significant.

Article Snippet: The plasmid of mouse STAT1 (BC004808) was purchased from Origene Technologies, Inc, USA.

Techniques: Transduction, Binding Assay, Purification, Western Blot, SDS Page, Cell Culture, Immunofluorescence, Microscopy, CCK-8 Assay, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Transcriptional inhibition of STAT1 functions in the nucleus alleviates Th1 and Th17 cell-mediated inflammatory diseases

doi: 10.3389/fimmu.2022.1054472

Figure Lengend Snippet: ndSTAT1-TMD inhibits the expression of STAT1-target genes at the transcription level. (A) HEK293T cells were co-transfected with the plasmids containing the luciferase reporter gene, whose expression is driven by the IL-17A promoter, and the wild-type STAT1 gene. The luciferase activity of cells was measured 24 hr after the treatment with ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) by a luminometer. Data are represented as mean ± SEM (n=5), and statistical significance was indicated when the luciferase activity was lower than the positive control group (pCMV-STAT1-only). The statistical analysis was examined using Student’s t-test. ****p<0.0001. (B, C) GSEA-based enrichment plots of the hallmark of IFN-γ response gene set in ndSTAT1-TMD-treated Th1 cells or of the gene set upregulated in STAT3 knockout CD4 + T cells compared to wild-type CD4 + T cells in ndSTAT1-TMD-treated Th17 cells. (D, E) Heatmap of Th1 or Th17 cell signature genes and naïve T cell-specific genes in Th1 or Th17 cells treated with or without ndSTAT1-TMD. Each column means results from one experimental well Z-score was calculated from log2(TPM+1).

Article Snippet: The plasmid of mouse STAT1 (BC004808) was purchased from Origene Technologies, Inc, USA.

Techniques: Expressing, Transfection, Luciferase, Activity Assay, Positive Control, Knock-Out

Journal: Bioorganic chemistry

Article Title: Bromo-substituted indirubins for inhibition of protein kinase-mediated signalling involved in inflammatory mediator release in human monocytes.

doi: 10.1016/j.bioorg.2024.107470

Figure Lengend Snippet: Fig. 3. Impact of indirubins 1 and 2 on the activation status of protein kinase pathways. A-F) Human monocytes were pre-treated with indirubins 1 or 2 at the indicated concentrations, or with inhibitors of GSK-3β (SB216763, 5 µM), CDK8 (CCT-251921, 40 nM), CDK9 (JSH-150, 20 nM) or CDK1 (Ro-3306, 10 µM) for 15 min prior to stimulation with LPS 100 ng/mL for 18 h. (A) Protein expression of β-catenin compared to GAPDH as housekeeping protein. (B) Levels of phosphorylated GSK-3β compared to total expression of GSK-3β protein. (C,D) Levels of phosphorylated STAT1 (C, Ser727 and D, Tyr701) vs. β-actin. (E) Levels of phosphorylated NF-κB (Ser276) compared to GAPDH. (F) Levels of phosphorylated Rb (Ser807/Ser811) vs. β-actin. Protein amounts were evaluated by Western Blot and densito metric analysis thereof. n = 3–4 biological replicates. Statistics: Data are shown as A-E) individual values and means ± SEM. Statistics were calculated by one-way ANOVA for multiple comparisons with Dunnett’s correction, testing treatments against vehicle (0.1 % (v/v) DMSO). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit monoclonal anti-COX-2, 1:1000 (12282, Cell Signaling, Danvers, MA), mouse monoclonal anti-β-catenin, 1:1000 (610153, BD Biosciences, San Jose, CA) mouse monoclonal anti-GSK-3β, 1:1000 (9832, Cell Signaling), rabbit monoclonal anti-phospho-GSK-3β (Ser9), 1:1000 (9323S, Cell Signaling), rabbit monoclonal anti-phosphoSTAT1 (Ser727), 1:1000 (ab109461, Abcam, Cambridge, UK), rabbit monoclonal anti-phospho-STAT1 (Tyr701), 1:1000 (ab109457, Abcam), rabbit monoclonal anti-phospho-NF-κB p65 (Ser276), 1:1000 (ab183559, Abcam), rabbit monoclonal anti-phospho-Rb (Ser807/811), 1:1000 (9308S, Cell Signaling), monoclonal anti-β-actin, 1:1000 (4970, Cell Signaling), mouse monoclonal anti-GAPDH, 1:1000 (97166S, Cell Signaling).

Techniques: Activation Assay, Expressing, Western Blot

Journal: Cell reports

Article Title: Tumor-specific CD4 T cells instruct monocyte fate in pancreatic ductal adenocarcinoma

doi: 10.1016/j.celrep.2023.112732

Figure Lengend Snippet: (A) NicheNet analysis from scRNA-seq data from using CD4 T cells as donor cells and MHCII hi macrophages as acceptor cells. See also . (B) Stat1 staining and MFI in MHCII hi and MHCII lo intratumoral macrophages at day 7. Data are mean ± SEM; n = 4 mice per group; ****p < 0.0001; Student’s t test. (C) Tumor weights from WT or Ifngr1 −/− mice; n = 4–8 mice per group; ****p < 0.0001; Student’s t test for each time point. (D) Proportion of MHCII hi or MHCII lo macrophages from WT or Ifngr1 −/− mice. Data are mean ± SEM; n = 4–8 mice per group; ****p < 0.0001; Student’s t test for each time point. (E) Proportion of Arg1 + macrophages on day 7. Data are mean ± SEM; ****p < 0.0001; Student’s t test. (F) Proportion of MHCII lo CD206 + FRβ + macrophages from WT or Ifngr1 −/− mice. Data are mean ± SEM; n = 4–8 mice per group; ****p < 0.0001; Student’s t test for each time point. (G) PD-L1 MFI gated on total macrophages from WT or Ifngr1 −/− mice. Data are mean ± SEM; **p < 0.005; Student’s t test for each time point. (H) Normalized expression of selected genes from bulk RNA sequencing from sorted live MHCII hi or MHCII lo tumor macrophages from WT and Ifngr1 −/− mice at day 14. See also . (I) Gene set enrichment analysis (GSEA) of the top 300 differentially expressed genes from sorted cells in (H). (J) Orthotopic KPC 2a tumor weights at day 7 from WT control mice, WT mice treated with CD40L blockade (αCD40L), or Cd40 −/− mice. *p < 0.05; one way ANOVA with Tukey’s post test. See also . (K) Proportion of MHCII hi or MHCII lo macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 4–5 mice per group; ***p < 0.001; one-way ANOVA with Tukey’s posttest. (L) Proportion of Arg1 + macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 4–5 mice per group; ***p < 0.001; one-way ANOVA with Tukey’s posttest. (M) Proportion of MHCII lo CD206 + FRβ + macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 4–5 mice per group; ***p < 0.001; one-way ANOVA with Tukey’s posttest. See also .

Article Snippet: anti-mouse Stat1 (clone D1K9Y) , Cell Signaling , Cat#: 80916S.

Techniques: Staining, Expressing, RNA Sequencing, Control

Journal: Cell reports

Article Title: Tumor-specific CD4 T cells instruct monocyte fate in pancreatic ductal adenocarcinoma

doi: 10.1016/j.celrep.2023.112732

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-mouse Stat1 (clone D1K9Y) , Cell Signaling , Cat#: 80916S.

Techniques: Recombinant, Red Blood Cell Lysis, Cell Isolation, Cell Stimulation, Software

Journal: eLife

Article Title: Tuberculosis-associated IFN-I induces Siglec-1 on tunneling nanotubes and favors HIV-1 spread in macrophages

doi: 10.7554/eLife.52535

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit monoclonal anti‐human STAT1 (clone 42H3) , Cell Signaling Technology , Cat# 9175 RRID: AB_2197984 , WB (1:100).

Techniques: Derivative Assay, Cell Culture, Transfection, Construct, Enzyme-linked Immunosorbent Assay, Blocking Assay, Control, Recombinant, Isolation, Western Blot, Fluorescence, Software, Imaging

Journal: Journal of Neuroinflammation

Article Title: The histone deacetylase inhibitor suberoylanilide hydroxamic acid attenuates human astrocyte neurotoxicity induced by interferon-γ

doi: 10.1186/1742-2094-9-113

Figure Lengend Snippet: Effects of SAHA on IFN-γ-induced phosphorylation of STAT1 and STAT3 in human astrocytes. Human astrocytes were incubated with or without SAHA at the concentrations indicated for 1 h. The cells were subsequently stimulated with IFN-γ for 30 minutes. Astrocytes in the control group were incubated with medium only. Cell lysates were separated by 8% SDS-PAGE and immunoblotted for phospho-Tyr 701 -STAT1 (pSTAT1) and total STAT1 (A) or phospho-Tyr 705 -STAT3 (pSTAT3) and total STAT3 (C) . The density ratios of phosphorylated to total protein are shown as mean ± S.E.M. of three independent experiments (B, D) . *Significantly different from IFN-γ stimulation only.

Article Snippet: The protein was transferred to a PVDF membrane at 70 V for 2 h. The membrane was blocked with 5% skim milk plus 3% bovine serum albumin (BSA) in PBS at room temperature (RT) for 1 h. Subsequently, the membrane was incubated with specific rabbit antibodies against phospho-Tyr 701 -STAT1 (1:2,000), total STAT1 (1:1,000), phospho-Tyr 705 -STAT3 (1:2,000) or total STAT3 (1:1,000) at 4°C overnight and then treated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2,000) at RT for 1 h. All antibodies used for immunoblotting were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Incubation, Control, SDS Page