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

Article Title: Opposing actions of Stat1 and Stat6 on IL-13-induced up-regulation of early growth response-1 and platelet-derived growth factor ligands in pulmonary fibroblasts.

doi: 10.4049/jimmunol.177.6.4141

Figure Lengend Snippet: FIGURE 3. Phosphorylation of Stat6 and Stat1 by IL-13 in mouse lung fibroblasts with Stat6 or Stat1 deletions. A, Western blot analysis of IL- 13-induced tyrosine phosphorylation of Stat1 (pStat1) in Stat6/ or wild- type Stat6/ fibroblasts. B, Western blot analysis of IL-13-induced ty- rosine phosphorylation of Stat6 (pStat6) in Stat1/ or wild-type Stat1/

Article Snippet: Egr-1 / mice and C57BL/6 (Egr-1 / ) mice, as well as Stat1 / mice and 129S6 (Stat1 / ) mice, were purchased from Taconic Farms.

Techniques: Phospho-proteomics, Western Blot

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

Article Title: Opposing actions of Stat1 and Stat6 on IL-13-induced up-regulation of early growth response-1 and platelet-derived growth factor ligands in pulmonary fibroblasts.

doi: 10.4049/jimmunol.177.6.4141

Figure Lengend Snippet: FIGURE 4. Suppression of IL-13-stimulated PDGF-A and PDGF-C mRNA expression by Stat1. RT-PCR of (A) PDGF-A or (B) PDGF-C mRNA expression in lung fibroblasts isolated from Stat1/ mice (F) or wild-type Stat1/ mice (E). PDGF-A and PDGF-C mRNA data were normalized to those of -actin and expressed as fold change relative to untreated control at time 0. Data are presented as mean values SD from triplicate samples and are representative of three independent experiments with similar results. , p 0.01; , p 0.001 compared with Stat1/.

Article Snippet: Egr-1 / mice and C57BL/6 (Egr-1 / ) mice, as well as Stat1 / mice and 129S6 (Stat1 / ) mice, were purchased from Taconic Farms.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Control

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

Article Title: Opposing actions of Stat1 and Stat6 on IL-13-induced up-regulation of early growth response-1 and platelet-derived growth factor ligands in pulmonary fibroblasts.

doi: 10.4049/jimmunol.177.6.4141

Figure Lengend Snippet: FIGURE 7. Enhanced IL-13-induced Egr-1 mRNA and protein expres- sion in Stat1/ lung fibroblasts. A, RT-PCR of IL-13-induced Egr-1 mRNA levels in lung fibroblasts isolated from Stat1/ mice (F) or wild- type Stat1/ mice (E). The Egr-1 mRNA data were normalized to those of -actin and expressed as fold change relative to untreated control at time 0. Data are presented as mean values SD from triplicate samples and are representative of three independent experiments with similar results. , p 0.01; , p 0.001 compared with Stat1/. B, Western blot analysis of IL-13-induced Egr-1 protein in Stat1/ or wild-type Stat1/ fibroblast cell lysates. C, Densitometry of IL-13 induced Egr-1 protein levels shown in B.

Article Snippet: Egr-1 / mice and C57BL/6 (Egr-1 / ) mice, as well as Stat1 / mice and 129S6 (Stat1 / ) mice, were purchased from Taconic Farms.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Control, Western Blot

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

Article Title: Opposing actions of Stat1 and Stat6 on IL-13-induced up-regulation of early growth response-1 and platelet-derived growth factor ligands in pulmonary fibroblasts.

doi: 10.4049/jimmunol.177.6.4141

Figure Lengend Snippet: FIGURE 10. Postulated mechanism of transcriptional regulation of PDGF ligand production by IL-13 in lung fibroblasts. IL-13 binds to re- ceptors on the lung fibroblast cell surface, which activate Stat6 and Stat1. Activation of the ERK1/2 pathway by IL-13 suppresses Stat6 signaling. The Stat transcription factors modulate Egr-1 expression that serves to regulate transcription of PDGF-A and PDGF-C and subsequent production of functional PDGF-AA and PDGF-CC dimeric proteins.

Article Snippet: Egr-1 / mice and C57BL/6 (Egr-1 / ) mice, as well as Stat1 / mice and 129S6 (Stat1 / ) mice, were purchased from Taconic Farms.

Techniques: Activation Assay, Expressing, Functional 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: 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: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: IFNγ treatment induces STAT1 activation in BV2 cells. BV2 cells were exposed to 20 ng/mL of IFNγ at different time points and activation of STAT1 was evaluated through different experiments. ( A ) Total protein extracts of IFNγ-treated BV2 cells at different time points were analyzed by Western Blot with anti-phospho-Tyr701 STAT1 antibody, and after stripping, the same blot was analyzed with anti-STAT1 antibody. Densitometric analysis revealed a STAT1 phosphorylation upon IFNγ treatment, peaking at 15 min compared with untreated BV2 cells (CTR). ( B ) RT-qPCR of iNOS and COX2, two STAT1-dependent genes, revealed an increase in their mRNA levels after 18 and 24 h of IFNγ treatment in BV2 cells. Data are expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 vs. untreated cells (CTR). Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Western Blot, Stripping Membranes, Phospho-proteomics, Quantitative RT-PCR

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: GEE pre-treatment counteracts IFNγ-dependent STAT1 activation in BV2 cells. ( A ) Total protein extracts from BV2 cells pre-treated overnight with GEE and subsequently exposed to 20 ng/mL IFNγ for 18 h were analyzed by Western Blot using a phospho-Tyr701 STAT1 antibody. The same blot was analyzed with anti-Tubulin antibody to check the amount of loaded proteins. Densitometric analysis showed decreased STAT1 phosphorylation in GEE pre-treated cells compared to CTR. * p < 0.05, ** p < 0.01 vs. untreated BV2 cells (CTR). # p < 0.05 vs. IFNγ-treated cells. ( B ) RT-qPCR analysis revealed that iNOS and COX2 mRNA levels were reduced in BV2 cells pre-treated overnight with GEE and then exposed to 20 ng/mL IFNγ for 24 h. Data are expressed as mean ± SD (n = 3). ** p < 0.01 vs. CTR, ## p < 0.01 vs. IFNγ-treated cells. Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Quantitative RT-PCR

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: GEE pre-treatment inhibits IFNγ-induced STAT1 phosphorylation and nuclear translocation in BV2 cells. The cells were immunostained with phospho-Tyr701 STAT1 (red) and analyzed by confocal microscopy (lens 60×). Nuclei were stained with DAPI (cyan). The orthogonal sections (ZX and ZY) of merge image reveal that pTYR 701 STAT1 protein is inside the nuclei. Scale bars indicate 10 μm. Images are representative of three separate experiments.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Phospho-proteomics, Translocation Assay, Confocal Microscopy, Staining

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: IFNγ treatment induces S-glutathionylation of STAT1. ( A ) Total protein extracts of BV2 cells left untreated (CTR) or treated with 20 ng/mL IFNγ for the indicated times were subjected to immunoprecipitation with anti-STAT1 antibody. Immunoprecipitated STAT1 (IP: STAT1) was analyzed by Western Blot under non-reducing conditions using anti-SSG antibody and, after membrane stripping, re-probed with anti-STAT1 antibody. Densitometric analysis revealed increased STAT1 S-glutathionylation after 5 and 15 min of IFNγ treatment compared to untreated BV2 cells. * p < 0.05, ** p < 0.01 vs. untreated BV2 cells (CTR). ( B ) Total protein lysates from the same untreated and treated BV2 cells were reserved before pull-down (input) and analyzed by Western Blot with anti-phospho-Tyr701 STAT1 antibody and, after membrane stripping, re-probed with anti-STAT1 antibody. Images are representative of four independent experiments. Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Immunoprecipitation, Western Blot, Membrane, Stripping Membranes

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: FNγ induces M1 polarization in BV2 cells through oxidative stress and STAT1 activation. ( A ) The expression of the pro-inflammatory cytokines IL-6 and TNFα in the supernatant of BV2 cells was evaluated by ELISA after 18 h of IFNγ stimulation, with or without GEE pretreatment. GEE markedly reduced the IFNγ-induced secretion of both cytokines, confirming the involvement of oxidative stress in M1 polarization. Data are expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 vs. CTR. # p < 0.05 vs. IFNγ. ( B ) Parental and STAT1 knockdown in BV2 cells were treated with 20 ng/mL of IFNγ for 18 h. Total protein extracts were analyzed by Western Blot with anti-iNOS and anti-Actin antibodies. Densitometric analysis revealed reduced iNOS induction in STAT1 knockdown in BV2 cells compared with parental cells upon IFNγ treatment. Parental and STAT1 knockdown in BV2 cells cultured without IFNγ were used as controls (CTR). Images are representative of three independent experiments. Single data points are shown as red dots. * p < 0.05, ** p < 0.01 vs. CTR. # p < 0.05 vs. IFNγ.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot, Cell Culture

Journal: iScience

Article Title: C9ORF72 suppresses JAK-STAT mediated inflammation

doi: 10.1016/j.isci.2023.106579

Figure Lengend Snippet:

Article Snippet: rabbit anti-pSTAT1 , R and D Systems , Cat# AF2894, RRID: AB_2198137.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Bradford Protein Assay, CRISPR, Plasmid Preparation, Software

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Baicalin suppresses the progression of Type 2 diabetes-induced liver tumor through regulating METTL3/m 6 A/HKDC1 axis and downstream p-JAK2/STAT1/clevaged Capase3 pathway.

doi: 10.1016/j.phymed.2021.153823

Figure Lengend Snippet: Fig. 4. The HKDC1 (m6A 2854 site) and HKDC1 protein levels in the HepG2 cells after treatment with siRNA fragments derived from m6A-related genes. A, The normalized level of HKDC1 (m6A 2854 site) treated with METTL3 siRNA. B, The normalized level of HKDC1 (m6A 2854 site) treated with METTL14 siRNA. C, The normalized level of HKDC1 (m6A 2854 site) treated with FTO siRNA. D, The normalized level of HKDC1 (m6A 2854 site) treated with ALKBH5 siRNA. E, The normalized level of HKDC1 (m6A 2854 site) treated with YTHDF1 siRNA. F, The protein bands of HKDC1/(p-)JAK2/(p-)STAT1/(cleaved-)caspase-3. G, The p-JAK/ JAK, p-STAT1/STAT1, and (cleaved-)caspase-3/caspase-3 ratios. The data are presented as mean ± SD, * p < 0.05 compared with the normal group, and & p < 0.05 compared with the high glucose + gene siRNA group (n = 3).

Article Snippet: The primary antibodies used in this study included p-JAK2, JAK2, p-STAT1, STAT1, cleaved-caspase-3, caspase-3, and β-actin (Santa Cruz Biotechnology, California, USA), anti-human HKDC1 antibody was purchased from Novus Biologicals Inc. (Colorado, USA).

Techniques: Derivative Assay

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Baicalin suppresses the progression of Type 2 diabetes-induced liver tumor through regulating METTL3/m 6 A/HKDC1 axis and downstream p-JAK2/STAT1/clevaged Capase3 pathway.

doi: 10.1016/j.phymed.2021.153823

Figure Lengend Snippet: Fig. 6. The protein levels of the p-JAK2/ STAT1/cleaved-caspase-3 pathway in the HepG2 cells at different glucose concentra tions. A, The western blot bands of the HKDC1 and p-JAK2/STAT1/cleaved-caspase-3 pathway. B, Statistical data of the protein expression levels. C, The p-JAK2/JAK2, p- STAT1/STAT1, and cleaved-caspase-3/caspase- 3 ratios. All data are presented as mean ± SD, * p < 0.05 compared with the normal group, and # p < 0.05 compared with the high glucose group (n = 3).

Article Snippet: The primary antibodies used in this study included p-JAK2, JAK2, p-STAT1, STAT1, cleaved-caspase-3, caspase-3, and β-actin (Santa Cruz Biotechnology, California, USA), anti-human HKDC1 antibody was purchased from Novus Biologicals Inc. (Colorado, USA).

Techniques: Western Blot, Expressing