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

Journal: Signal Transduction and Targeted Therapy

Article Title: Bioengineered iPSC-derived human macrophages with increased angiotensin-converting enzyme (ACE) expression suppress solid tumor growth

doi: 10.1038/s41392-026-02650-3

Figure Lengend Snippet: Determination of M1 vs. M2 polarization in iMac. a Pro-inflammatory M1 cytokines IL-1β, TNFα, IL-6, and IL-8 measured by ELISA. b Anti-inflammatory M2 cytokines IL-4, IL-10, IL-13, CCL5, CCL17, and CCL20 measured by ELISA. For a , b iMac were cultured in 6-well plates (3 × 10 6 cells/well). To challenge with tumor factors, iMac were co-cultured with 50% fresh supernatant from SK-MEL-28 cells every 24 h for 3 days. Supernatants were collected during the final 24 h for cytokine measurement. c Activation (phosphorylation) of NF-kB p65, STAT1, STAT3, and STAT6 in iMac was determined using Western blotting after conditioning with melanoma (SK-MEL-28) supernatant for 72 h. Protein band intensity was quantified using Odyssey version 3.0 software. After adjusting for loading using the β-actin band, data are presented as fold change to the iMac (Dox-) group, which is set as 1. For a – c , one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SD. Each experiment was performed three times, with triplicate samples each time. * p < 0.05, *** p < 0.001, and **** p < 0.0001

Article Snippet: The polyvinylidene difluoride (PVDF) membranes were incubated with specific primary antibodies against ACE (R&D Systems, MAB9291, 1:1,000), GAPDH (Sigma-Aldrich, SAB5600208, 1:2,000), β-actin (Sigma-Aldrich, A3854; 1:1000), phosphorylated NF-kB p65 (Novus, NB100-82086, 1:500), phosphorylated STAT1 (R&D Systems, AF2894, 1 μg/ml), phosphorylated STAT3 (Novus, NBP2-24463, 0.5 μg/ml), or phosphorylated STAT6 (Millipore, 06-937, 1:1000).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, Phospho-proteomics, Western Blot, Software

Journal: Immunology

Article Title: Interferon-α promotes MHC I antigen presentation of islet β cells through STAT1-IRF7 pathway in type 1 diabetes.

doi: 10.1111/imm.13468

Figure Lengend Snippet: FIGURE 5 Differential gene expression in NOD mice pancreas after IFN-α treatment. Representative images (40×) of frozen sections of mice pancreas stained for Insulin, DAPI and (a) STAT1, (b) IRF7, (c) TAP1, (d) MHC I from NOD mice with or without IFN-α treatment

Article Snippet: Phospho- STAT1, STAT1, Phospho- STAT2, STAT2, TAP1 and β- actin antibodies were purchased from Cell Signaling Technology company, USA; IRF7 antibody was purchased from NOVUS company, USA; B2M, PSMB8 and NLRC5 antibodies were purchased from Abcam company, UK; PhosphoSTAT3, STAT3 and Phospho- STAT5 antibodies were purchased from Abclonal company, China; STAT5 and IRF9 antibodies were purchased from company, China.

Techniques: Gene Expression, Staining

Journal: Immunology

Article Title: Interferon-α promotes MHC I antigen presentation of islet β cells through STAT1-IRF7 pathway in type 1 diabetes.

doi: 10.1111/imm.13468

Figure Lengend Snippet: FIGURE 6 Inhibition of STAT1 and STAT2 prevented IFN-α induced MHC-I and antigen presentation in MIN6 cells. MIN6 cells were transfected with siRNA (si-STAT1 or/and si-STAT2) and then treated with or without IFN-α (1000 U/ml) for 48 h. (a) Protein and (b) mRNA of antigen presented molecules was measured. (c) Representative images (10×) of MHC I in MIN6 cells after IFN-α and siRNA treatment. *p < 0·05, **p < 0·01 and ***p < 0·001

Article Snippet: Phospho- STAT1, STAT1, Phospho- STAT2, STAT2, TAP1 and β- actin antibodies were purchased from Cell Signaling Technology company, USA; IRF7 antibody was purchased from NOVUS company, USA; B2M, PSMB8 and NLRC5 antibodies were purchased from Abcam company, UK; PhosphoSTAT3, STAT3 and Phospho- STAT5 antibodies were purchased from Abclonal company, China; STAT5 and IRF9 antibodies were purchased from company, China.

Techniques: Inhibition, Immunopeptidomics, Transfection

Journal: Immunology

Article Title: Interferon-α promotes MHC I antigen presentation of islet β cells through STAT1-IRF7 pathway in type 1 diabetes.

doi: 10.1111/imm.13468

Figure Lengend Snippet: FIGURE 8 IFN-α induce β cells autoimmunity by promoting MHC I antigen presentation. In β cells, IFN-α binds to the IFNRs and lead to the nuclear translocation of STAT1 and IRF7, which in turn activates the STAT1-IRF7 axis to increase expression of antigen presenting molecules (TAP1, PSMB8 and MHC I). These actions create positive feedback through IRF7-STAT2 cascade amplifying signals and promote the proliferation of CD8+ T cells and insulitis. Figure was created with BioRender.com (Agreement number: TX23NA2XMH)

Article Snippet: Phospho- STAT1, STAT1, Phospho- STAT2, STAT2, TAP1 and β- actin antibodies were purchased from Cell Signaling Technology company, USA; IRF7 antibody was purchased from NOVUS company, USA; B2M, PSMB8 and NLRC5 antibodies were purchased from Abcam company, UK; PhosphoSTAT3, STAT3 and Phospho- STAT5 antibodies were purchased from Abclonal company, China; STAT5 and IRF9 antibodies were purchased from company, China.

Techniques: Immunopeptidomics, Translocation Assay, Expressing

Journal: Journal of clinical immunology

Article Title: Hematopoietic Stem Cell Transplantation in Patients with Heterozygous STAT1 Gain-of-Function Mutation

doi: 10.1007/s10875-018-0575-y

Figure Lengend Snippet: (A) Pre- and post-transplantation total p-STAT1 expression in CD4+ T cells stimulated with IFN-β (20 ng/mL) by flow cytometry in patient (P1) compared to healthy control. (B) The dose response curve of STAT1 phosphorylation induced with IFN-β in patient and control CD4+ T cells. *** p<0.0001 by two-way ANOVA. Tx: Transplantation.

Article Snippet: Monoclonal antibodies (mAbs) to the following human proteins were used for staining: CD3 (UCHT1), CD4 (RPA-T4), IFN-γ (4S.B3), IL-17 (BL168) (Biolegend), phospho (p)-STAT1 (KIKSI0803), (all from eBioscience), STAT1 (246523; R&D Systems).

Techniques: Transplantation Assay, Expressing, Flow Cytometry, Control, Phospho-proteomics

Journal: Journal of clinical immunology

Article Title: Hematopoietic Stem Cell Transplantation in Patients with Heterozygous STAT1 Gain-of-Function Mutation

doi: 10.1007/s10875-018-0575-y

Figure Lengend Snippet: The details and outcome of HSCT in this study and other studies for STAT1 GOF mutated patients

Article Snippet: Monoclonal antibodies (mAbs) to the following human proteins were used for staining: CD3 (UCHT1), CD4 (RPA-T4), IFN-γ (4S.B3), IL-17 (BL168) (Biolegend), phospho (p)-STAT1 (KIKSI0803), (all from eBioscience), STAT1 (246523; R&D Systems).

Techniques: Mutagenesis, Infection