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

Article Title: USP39 Serves as a Deubiquitinase to Stabilize STAT1 and Sustains Type I IFN-Induced Antiviral Immunity.

doi: 10.4049/jimmunol.1901384

Figure Lengend Snippet: FIGURE 3. USP39 promotes type I IFN antiviral signaling. (A) 293T cells were transfected with either control siRNA or siUSP39, together with ISRE- Luc and Renilla luciferase reporter plasmid for 48 h. The luciferase activity was measured after further 12 h IFN-a (1000 IU/ml) treatment. (B) 293T cells were transfected with either vector or Flag-USP39, together with ISRE-Luc and Renilla for 48 h. The luciferase activity was measured after further 12 h of IFN-a (1000 IU/ml) treatment. (C) 293T cells were transfected with either control siRNA or siUSP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h, and mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (D) 293T cells were transfected with either vector or Flag-USP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h. The mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (E) U3A (STAT1 deficient) or its parental cells, HT1080, were transfected with either control siRNA or siUSP39. After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. The knockdown efficiency of USP39 was analyzed by Western blot. Viral levels were analyzed by flow cytometry. Data are from three experiments and are mean and SD of three independent replicates. (A–E, right panels) or representative of three independent experiments (E left panel). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. A p value .0.05 was considered ns.

Article Snippet: The following Abs were used: Abs against USP39 (1:2000; ab131244; Abcam, Cambridge, MA), IFNR1 (1:1000; DF6571; Affinity, Cincinnati, OH), IFNR2 (1:1000; sc-271105; Santa Cruz Biotechnology, Santa Cruz, CA), STAT1 (1:1000; catalog no. 9172; Cell Signaling Technology [CST], Danvers, MA), pY701-STAT1 (1:1000; catalog no. 9167; CST), STAT2 (1:1000; catalog no. 72704; CST), pY690-STAT2 (1:1000; catalog no. 88410; CST), JAK1 (1:1000; sc-1677; Santa Cruz Biotechnology), pJAK1 (1:1000; catalog no. 3331s; CST), Tyk2 (1:1000; catalog no. 14193; CST), p-Tyk2 (1:1000; catalog no. 9321; CST), VSV-G (1:5000; sc-66180; Santa Cruz Biotechnology), HA (1:2000; ab9110; Abcam), ubiquitin (1:1000; sc-8017; Santa Cruz Biotechnology), Flag (1:2500; F7425; Sigma-Aldrich), Myc (1:2000; catalog no. 9B11; CST), GAPDH (1:20,000; G9545; Sigma-Aldrich), tubulin (1:5000; 66031-1-Ig; Proteintech, Rosemont, IL).

Techniques: Transfection, Control, Luciferase, Plasmid Preparation, Activity Assay, Real-time Polymerase Chain Reaction, Infection, Knockdown, Western Blot, Cytometry

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

Article Title: USP39 Serves as a Deubiquitinase to Stabilize STAT1 and Sustains Type I IFN-Induced Antiviral Immunity.

doi: 10.4049/jimmunol.1901384

Figure Lengend Snippet: FIGURE 4. USP39 interacts with STAT1. (A) 293T cells were transfected with either control siRNA or siUSP39, and mRNA levels of type I IFN molecules were measured by real-time PCR. (B) 293T cells transfected with either control siRNA (con siRNA) or siUSP39 were stimulated with IFN-a (1000 IU/ml) for 0, 30, and 60 min. Cell lysates were analyzed by immunoblotting as indicated. (C) 293T cells were transfected with either vector or Flag- USP39. After 48 h, the protein level of STAT1 was measured by Western blot. (D) 293T cells were transfected with Flag-USP39 plasmid. Forty-eight hours posttransfection, the cells were lysed and immunoprecipitated by STAT1 Ab and protein A agarose beads, The USP39 was analyzed using anti-Flag Ab. Data are from three experiments are mean and SD of three independent replicates (A) or representative of three independent experiments (B–D). A p value .0.05 was considered ns.

Article Snippet: The following Abs were used: Abs against USP39 (1:2000; ab131244; Abcam, Cambridge, MA), IFNR1 (1:1000; DF6571; Affinity, Cincinnati, OH), IFNR2 (1:1000; sc-271105; Santa Cruz Biotechnology, Santa Cruz, CA), STAT1 (1:1000; catalog no. 9172; Cell Signaling Technology [CST], Danvers, MA), pY701-STAT1 (1:1000; catalog no. 9167; CST), STAT2 (1:1000; catalog no. 72704; CST), pY690-STAT2 (1:1000; catalog no. 88410; CST), JAK1 (1:1000; sc-1677; Santa Cruz Biotechnology), pJAK1 (1:1000; catalog no. 3331s; CST), Tyk2 (1:1000; catalog no. 14193; CST), p-Tyk2 (1:1000; catalog no. 9321; CST), VSV-G (1:5000; sc-66180; Santa Cruz Biotechnology), HA (1:2000; ab9110; Abcam), ubiquitin (1:1000; sc-8017; Santa Cruz Biotechnology), Flag (1:2500; F7425; Sigma-Aldrich), Myc (1:2000; catalog no. 9B11; CST), GAPDH (1:20,000; G9545; Sigma-Aldrich), tubulin (1:5000; 66031-1-Ig; Proteintech, Rosemont, IL).

Techniques: Transfection, Control, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Immunoprecipitation

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

Article Title: USP39 Serves as a Deubiquitinase to Stabilize STAT1 and Sustains Type I IFN-Induced Antiviral Immunity.

doi: 10.4049/jimmunol.1901384

Figure Lengend Snippet: FIGURE 5. USP39 stabilizes STAT1 protein level. (A) 293T cells were transfected with either vector or Flag-USP39 and then treated with CHX (50 mg/ml) for the indicated times. Cell lysates were analyzed by immunoblotting as indicated. (B) 293T cells were transfected with either control siRNA or siUSP39, together with HA-Ub plasmids. STAT1 proteins were immunoprecipitated, and STAT1 ubiquitination levels were analyzed. (C) 293T cells were cotransfected with Flag-STAT1 and HA-Ub or Flag-USP39 expression plasmids, respectively. Forty-eight hours posttransfection, the Flag-STAT1 and Flag- USP39 proteins were purified from 293T cells by anti-Flag beads. Both immunoprecipitated proteins were incubated at 37˚C for 2 h with deubiquitination buffer, followed by immunoblotting as indicated for in vitro deubiquitination assay. (D) Myc-STAT1 proteins were purified from 293T cells cotransfected with Myc-STAT1 and HA-Ub by anti-Myc beads and then incubated with Flag-USP39 or Flag-USP39 (C136/139A) mutant proteins immunoprecipitated by anti-Flag beads in vitro at 37˚C for 2 h with deubiquitination buffer, followed by immunoblotting as indicated. (E) 293T cells were transfected with vector, Flag-USP39, and Flag-USP39 (C136/C139A). After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. Viral level, IFN-b, IFIT1, and ISG54 mRNA levels were analyzed by real-time PCR. **p , 0.01, ***p , 0.001. A p value . 0.05 was considered ns.

Article Snippet: The following Abs were used: Abs against USP39 (1:2000; ab131244; Abcam, Cambridge, MA), IFNR1 (1:1000; DF6571; Affinity, Cincinnati, OH), IFNR2 (1:1000; sc-271105; Santa Cruz Biotechnology, Santa Cruz, CA), STAT1 (1:1000; catalog no. 9172; Cell Signaling Technology [CST], Danvers, MA), pY701-STAT1 (1:1000; catalog no. 9167; CST), STAT2 (1:1000; catalog no. 72704; CST), pY690-STAT2 (1:1000; catalog no. 88410; CST), JAK1 (1:1000; sc-1677; Santa Cruz Biotechnology), pJAK1 (1:1000; catalog no. 3331s; CST), Tyk2 (1:1000; catalog no. 14193; CST), p-Tyk2 (1:1000; catalog no. 9321; CST), VSV-G (1:5000; sc-66180; Santa Cruz Biotechnology), HA (1:2000; ab9110; Abcam), ubiquitin (1:1000; sc-8017; Santa Cruz Biotechnology), Flag (1:2500; F7425; Sigma-Aldrich), Myc (1:2000; catalog no. 9B11; CST), GAPDH (1:20,000; G9545; Sigma-Aldrich), tubulin (1:5000; 66031-1-Ig; Proteintech, Rosemont, IL).

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Immunoprecipitation, Ubiquitin Proteomics, Expressing, Incubation, In Vitro, Mutagenesis, Infection, Real-time Polymerase Chain Reaction

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

Article Title: USP39 Serves as a Deubiquitinase to Stabilize STAT1 and Sustains Type I IFN-Induced Antiviral Immunity.

doi: 10.4049/jimmunol.1901384

Figure Lengend Snippet: FIGURE 6. USP39 decreases K6-linked ubiquitination of STAT1. (A) 293T cells were transfected with Flag- STAT1, together with HA-Ub-K6, HA-Ub-K11, HA- Ub-K27, HA-Ub-K29, HA-Ub-K33, HA-Ub-K48 or HA-Ub-K63, and siUSP39. Immunoprecipitation was performed using anti-Flag M2 beads, followed by im- munoblotting using an anti-HA Ab. (B) 293T cells seeded in the 6-cm dish were transfected with HA vector or HA-K6, together with Flag-STAT1 plasmids. Thirty-six hours posttransfection, cells were treated with DMSO or MG132 (10 mM) for 6 and 12 h. Cell lysates were incubated with anti-Flag beads at 4˚C overnight, and the K6-linked ubiquitination of STAT1 was analyzed by Western blot. Data are representative of three independent experiments.

Article Snippet: The following Abs were used: Abs against USP39 (1:2000; ab131244; Abcam, Cambridge, MA), IFNR1 (1:1000; DF6571; Affinity, Cincinnati, OH), IFNR2 (1:1000; sc-271105; Santa Cruz Biotechnology, Santa Cruz, CA), STAT1 (1:1000; catalog no. 9172; Cell Signaling Technology [CST], Danvers, MA), pY701-STAT1 (1:1000; catalog no. 9167; CST), STAT2 (1:1000; catalog no. 72704; CST), pY690-STAT2 (1:1000; catalog no. 88410; CST), JAK1 (1:1000; sc-1677; Santa Cruz Biotechnology), pJAK1 (1:1000; catalog no. 3331s; CST), Tyk2 (1:1000; catalog no. 14193; CST), p-Tyk2 (1:1000; catalog no. 9321; CST), VSV-G (1:5000; sc-66180; Santa Cruz Biotechnology), HA (1:2000; ab9110; Abcam), ubiquitin (1:1000; sc-8017; Santa Cruz Biotechnology), Flag (1:2500; F7425; Sigma-Aldrich), Myc (1:2000; catalog no. 9B11; CST), GAPDH (1:20,000; G9545; Sigma-Aldrich), tubulin (1:5000; 66031-1-Ig; Proteintech, Rosemont, IL).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Plasmid Preparation, Incubation, Western Blot

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

Article Title: USP39 Serves as a Deubiquitinase to Stabilize STAT1 and Sustains Type I IFN-Induced Antiviral Immunity.

doi: 10.4049/jimmunol.1901384

Figure Lengend Snippet: FIGURE 8. A proposed model of the regulation of STAT1 by USP39 through ubiquitin proteasome system. Without viral infection, the cytoplasmic STAT1 protein level was regulated by USP13 to reduce the STAT1 ubiquitination or by Smurf1 and SLIM to induce the ubiquitination of STAT1, which sustains the STAT1 protein level. USP39 is required for the stable association of the U4/U6·U5 trisnRNP to the prespliceosome, which is essential for pre- mRNA splicing. USP39 can promote tumor growth through the Wnt/b-catenin pathway or the AKT signaling pathway, etc. When the cells are infected with viruses, USP39 binds to nuclear STAT1 and cleaves K6-linked ubiquitin chains of STAT1 and therefore sustains STAT1 levels in the nucleus. STAT1 exports from the nucleus to the cytoplasm. IFNs promote the phosphorylation of STAT1 and the nuclear import of p-STAT1; ISGF3 binds to ISRE promoter to induce the expression of ISGs. Finally, these ISGs perform antiviral function.

Article Snippet: The following Abs were used: Abs against USP39 (1:2000; ab131244; Abcam, Cambridge, MA), IFNR1 (1:1000; DF6571; Affinity, Cincinnati, OH), IFNR2 (1:1000; sc-271105; Santa Cruz Biotechnology, Santa Cruz, CA), STAT1 (1:1000; catalog no. 9172; Cell Signaling Technology [CST], Danvers, MA), pY701-STAT1 (1:1000; catalog no. 9167; CST), STAT2 (1:1000; catalog no. 72704; CST), pY690-STAT2 (1:1000; catalog no. 88410; CST), JAK1 (1:1000; sc-1677; Santa Cruz Biotechnology), pJAK1 (1:1000; catalog no. 3331s; CST), Tyk2 (1:1000; catalog no. 14193; CST), p-Tyk2 (1:1000; catalog no. 9321; CST), VSV-G (1:5000; sc-66180; Santa Cruz Biotechnology), HA (1:2000; ab9110; Abcam), ubiquitin (1:1000; sc-8017; Santa Cruz Biotechnology), Flag (1:2500; F7425; Sigma-Aldrich), Myc (1:2000; catalog no. 9B11; CST), GAPDH (1:20,000; G9545; Sigma-Aldrich), tubulin (1:5000; 66031-1-Ig; Proteintech, Rosemont, IL).

Techniques: Ubiquitin Proteomics, Infection, Phospho-proteomics, Expressing

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: (A) IFN-β alone vs media, expressed as ratio of IFN-β/media. (B) Vitamin D alone vs media. (C) IFN-β plus vitamin D vs media alone. Selected targets are grouped into Th1, Th17, and Th2 immune pathways and clusters of IFN-stimulated proteins (ISGs), adhesion molecules, and cytokines controlling homeostatic proliferation, neurotrophic factors, and IL-12 family. Values are fold change in the stimulation condition/media. Intensity of shading shows upregulation (red) or downregulation (blue). Target proteins include BDNF = brain-derived neurotrophic factor; HGF = hepatocyte growth factor; IFN-γ = interferon-γ; IL-2 = interleukin-2; TNF-α = tumor necrosis factor-α; TNFRII = TNF receptor type II; IL-17F, IL-4, IL-5, IL-10, IP-10 = IFN-induced protein-10 (CXCL10); MCP1 = macrophage chemotactic protein-1 (CCL2); I-TAC = IFN-inducible T-cell alpha chemoattractant (CXCL11); LIF = leukemia inhibitory factor; p-Y-STAT1 = phosphotyrosine-STAT1 transcription factor; MxA = myxovirus A; NGF = nerve growth factor; sICAM-1 = soluble intercellular adhesion molecule-1; TPO = thymopoietin; VCAM-1 = vascular cell adhesion molecule; VEGF-α = vascular endothelial growth factor-α, IL-15, IL-7, and IL-12 p40 and IL-12 p70 components.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Derivative Assay

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: (A) In vitro vitamin D enhances IFN-β induction of p-Y-STAT1 in ConA-activated MNCs from 72 untreated patients with MS ( p = 0.00002). (B) p-Y-STAT1 expression on flow cytometry histograms from a representative patient with RRMS-s. The media, IFN, VitD, and IFN+VitD curves are all stained with Alexa Fluor 488–labeled Mab to p-Y-STAT1. (C) MxA protein on Western blot of a representative patient with RRMS-s. ConA-activated MNCs were incubated in vitro with 160 U/mL IFN-β-1b for 30 minutes to 48 hours ± preincubation with 200 nM Vit D3 (calcitriol) for 12 hours. Gray-scale densities of each band are listed above figure. IFN-β = interferon-β; MxA = myxovirus protein.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: In Vitro, Expressing, Flow Cytometry, Staining, Labeling, Western Blot, Incubation

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: (A) p-Y-STAT1, measured with flow cytometry, is generated in all groups, but there is less induction in SPMS than in HC. (B) MxA protein, measured with Western blots. The trend for enhanced responses in PPMS vs HC was not significant ( p < 0.13, unpaired t test). ConA-activated MNCs were incubated in vitro with 160 U/mL IFN-β-1b for 30 minutes to 48 hours ± preincubation with 200 nM vitamin D3 (calcitriol) for 12 hours. Fold change of vitamin D plus IFN-β compared with IFN-β alone, determined using VDSI: [(IFN + VitD) − (VitD)]/[(IFN) − (no IFN)]; average with p values above SEM bar: * p < 0.05, ** p < 0.01. Comparisons between groups use unpaired t tests, brackets. PPMS = primary progressive MS; RRMS-a = active relapsing/remitting MS; RRMS-s = stable RRMS; SPMS = secondary progressive MS.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Generated, Western Blot, Incubation, In Vitro

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: In all groups, there is enhanced induction of p-Y-STAT1, measured with flow cytometry. Methods and calculations as in . * p < 0.05, ** p < 0.01. IFN-β = interferon-β; RRMS-a = active relapsing/remitting MS; RRMS-s = stable RRMS; SPMS = secondary progressive MS.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Flow Cytometry

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: Vitamin D plus IFN-β–induced p-Y-STAT1 expression is quantitated with flow cytometry. The median vitamin D level for the entire cohort was 30; low < 30, high ≥ 30 ng/mL 25-OH vitamin D. Methods and calculations as in . * p < 0.05, ** p < 0.01. RRMS-a = active relapsing/remitting MS; RRMS-s = stable RRMS; PPMS = primary progressive MS; SPMS = secondary progressive MS.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: Vitamin D plus IFN-β (blue line) in most therapy-naive groups had a greater effect than IFN-β alone (yellow line) on activation of STAT1 in ConA-activated lymphocytes and monocytes. Values in radar plot represent median fold change in log 2 scale expression of p-Y-STAT1 measured by flow cytometry. Blue line: (IFN+ VitD) − (Vit D); yellow: (IFN)-(media alone). IFN-β = interferon-β; RRMS-a = active relapsing/remitting MS; RRMS-s = stable RRMS; SPMS = secondary progressive MS.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Activation Assay, Expressing, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Differentiation status determines the effects of IFNγ on the expression of PD-L1 and immunomodulatory genes in melanoma

doi: 10.1186/s12964-024-01963-6

Figure Lengend Snippet: IFNγ-induced PD-L1 expression in 624Mel cells is dependent on JAK kinase activity irrespective of differentiation. A , B , C Quantitative RT-PCR of MITF, IRF1 and PD-L1 mRNA expression in 624Mel cells transfected with control siRNA (siCTRL) or siMITF, with or without 5 ng/mL IFNγ and/or 300 nM JAK inhibitor. D , E , F Western blotting of STAT1, pSTAT1 (Tyr701) and β-Actin in 624Mel cells transfected with siCTRL or siMITF, with or without 5 ng/mL IFNγ and/or 900 nM JAK inhibitor. G , H , I , J Western blotting of MITF, IRF1, PD-L1 and β-Actin in 624Mel cells transfected with siCTRL or siMITF, with or without 5 ng/mL IFNγ and/or 900 nM JAK inhibitor. Plots indicate mean ± standard deviations of value distributions. Statistical analysis performed by one-way ANOVA and Tukey’s multiple comparison test, adjusted P value ** = < 0.01, *** = < 0.001, **** = < 0.0001 ( n = 5 for A-C, n = 4 for E-J)

Article Snippet: The antibodies used in this study include: Anti-actin (clone C4, Millipore, MAB1501, 1:5000), β-actin (D6A8, Cell Signaling, #8457, 1:2500), IRF-1 (D5E4, Cell Signaling, #8478, 1:1000), PD-L1 (E1L3N, Cell Signaling, #13,684, 1:1000), Stat1 (D1K9Y, Cell Signaling, #14,994, 1:1000), p-Stat1 (Y701) (D4A7, Cell Signaling, #7649, 1:1000) and Anti-Microphthalmia (clone c5, Millipore, MAB3747, 1:500).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Transfection, Control, Western Blot, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Differentiation status determines the effects of IFNγ on the expression of PD-L1 and immunomodulatory genes in melanoma

doi: 10.1186/s12964-024-01963-6

Figure Lengend Snippet: IFNγ-induced PD-L1 expression is dependent on STAT1, irrespective of differentiation status. A , B , C , D Quantitative RT-PCR of MITF, STAT1, IRF1 and PD-L1 mRNA expression in 624Mel cells transfected with siCTRL, siMITF, siSTAT1 or both siMITF and siSTAT1, with or without 5 ng/mL IFNγ. E , F , G , H , I Western blotting of MITF, STAT1, IRF1, PD-L1 and β-Actin in 624Mel cells transfected with siCTRL, siMITF, siSTAT1 or both siMITF and siSTAT1 with or without 5 ng/mL IFNγ. Plots indicate mean ± standard deviations of value distributions. Statistical analysis performed by one-way ANOVA and Tukey’s multiple comparisons test, adjusted P value ** = < 0.01, *** = < 0.001, **** = < 0.0001 (n = 3)

Article Snippet: The antibodies used in this study include: Anti-actin (clone C4, Millipore, MAB1501, 1:5000), β-actin (D6A8, Cell Signaling, #8457, 1:2500), IRF-1 (D5E4, Cell Signaling, #8478, 1:1000), PD-L1 (E1L3N, Cell Signaling, #13,684, 1:1000), Stat1 (D1K9Y, Cell Signaling, #14,994, 1:1000), p-Stat1 (Y701) (D4A7, Cell Signaling, #7649, 1:1000) and Anti-Microphthalmia (clone c5, Millipore, MAB3747, 1:500).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Differentiation status determines the effects of IFNγ on the expression of PD-L1 and immunomodulatory genes in melanoma

doi: 10.1186/s12964-024-01963-6

Figure Lengend Snippet: IFNγ-induced PD-L1 expression in dedifferentiated melanoma cells is dependent on IRF1 . A , B , C Quantitative RT-PCR of MITF, IRF1 and PD-L1 mRNA expression in 624Mel cells transfected with Control siRNA (siCTRL), siMITF, siIRF1 or both siMITF and siIRF1, with or without 5 ng/mL IFNγ. E , F , G Western blotting of MITF, STAT1, IRF1, PD-L1 and β-Actin in 624Mel cells transfected with siCTRL, siMITF, siIRF1 or both siMITF and siIRF1, with or without 5 ng/mL IFNγ. Plots indicate mean ± standard deviations of value distributions. Statistical analysis performed by one-way ANOVA and Tukey’s multiple comparisons test, adjusted P value * = < 0.05, ** = < 0.01, *** = < 0.001, **** = < 0.0001 ( n = 4 for A-C, n = 5 for D-G)

Article Snippet: The antibodies used in this study include: Anti-actin (clone C4, Millipore, MAB1501, 1:5000), β-actin (D6A8, Cell Signaling, #8457, 1:2500), IRF-1 (D5E4, Cell Signaling, #8478, 1:1000), PD-L1 (E1L3N, Cell Signaling, #13,684, 1:1000), Stat1 (D1K9Y, Cell Signaling, #14,994, 1:1000), p-Stat1 (Y701) (D4A7, Cell Signaling, #7649, 1:1000) and Anti-Microphthalmia (clone c5, Millipore, MAB3747, 1:500).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Western Blot

Journal: PLoS ONE

Article Title: Lithium Controls Central Nervous System Autoimmunity through Modulation of IFN-γ Signaling

doi: 10.1371/journal.pone.0052658

Figure Lengend Snippet: (A and B) Splenocytes were pre-incubated for 1 h in the absence or presence of the GSK3 inhibitors LiCl or TDZD-8, and stimulated without or with IFN-γ (5 U/ml; 25 minutes) or IFN-β (100 U/ml; 45 minutes) and/or anti-CD3 (1.25 µg/ml; 25 minutes in (A), and 45 minutes in (B) as indicated. Mononuclear cells were stained for pSTAT1-Y701 and analyzed by flow cytometry. Representative histograms are gated on CD4 + T cells. Induction of pSTAT1-Y701 is normalized to unstimulated cells. Fold induction of pSTAT1-Y701 MFI is normalized to unstimulated cells from combined data of 2-4 experiments. * p <0.05, as determined by one-way ANOVA. (C) Thioglycollate-elicited macrophages (left histogram) were pre-incubated for 1 h in the absence or presence LiCl. Cells were then stimulated for 25 minutes with IFN-γ (5 U/ml) or left unstimulated, stained and analyzed for pSTAT1-Y701 in CD11b + gated cells as in (A). CD11b + cells (right histogram) were isolated from dLNs and spleens of MOG 35–55 -immunized mice, restimulated for 24 h with MOG 35–55 (10 µg/ml) in the absence or presence of LiCl and evaluated for pSTAT1-Y701. (D) Naïve splenocytes from Ifngr1 −/− mice were pre-incubated without or with LiCl, and stimulated for 25 minutes with IFN-γ (5 U/ml) and/or αCD3 (1.25 µg/ml), as indicated, and evaluated for pSTAT1-Y701. Histograms are gated on CD4 + T cells.

Article Snippet: Cells were stained with anti-CD4 (RM4-5; Biolegend), fixed, permeabilized with Phosflow Perm Buffer III (BD Pharmingen) and stained with an antibody against phospho-STAT1-Y701 (p-STAT1) (58D6, Cell Signaling).

Techniques: Incubation, Staining, Flow Cytometry, Isolation

Journal: PLoS ONE

Article Title: Lithium Controls Central Nervous System Autoimmunity through Modulation of IFN-γ Signaling

doi: 10.1371/journal.pone.0052658

Figure Lengend Snippet: (A and B) Cells from dLNs and spleen of MOG 35–55 immunized mice (10–21 d post immunization) were either (A) pre-incubated without or with LiCl, and left unstimulated or stimulated for 25 with IFN-γ (5 U/ml) and/or anti-CD3 (1.25 µg/ml) as indicated; or (B) restimulated for 24 h with MOG 35–55 (10 µg/ml) in the absence or presence of LiCl, from onset, or acutely treated for 1 h, as indicated. A subset of cells was stimulated after 24 h with IFN-γ (5 U/ml) for 25 minutes. Cells were gated on CD4 + T cells and pSTAT1-Y701 was analyzed as in Fig. 2 and normalized to unstimulated cells from naïve mice. Results are expressed as percent of control, which represent stimulated untreated samples (100%), n = 3. (C) IFN-γ production by 24 h MOG 35–55 restimulated cells (from B). Representative sample shown ( n = 3). (D) CD4 + T cells from spleens and dLNs of MOG 35–55 immunized mice were polarized under Th1 conditions in the presence or absence of LiCl, from onset or acutely treated for 1 h on day 3. Where indicated cells were stimulated with IFN-γ on day 3. Cells were gated on in CD4 + , analyzed for pSTAT1-Y701, and normalized to unstimulated cells from naïve mice. Results are expressed as percent of control, which represent stimulated untreated samples (100%), ( n = 3) (E), Th1 cells generated by polarization in the absence or presence of LiCl. Dot plots reflect CD4 + gated T cells. Representative experiment shown ( n = 2). (F) IFN-γ production from Th1 cells (day 3 of polarization) stimulated with anti-CD3 and anti-CD28 (1 µg/ml each) for 8 h in the absence or presence of LiCl was assessed by ELISA. Representative results shown ( n = 2). * p <0.05, as determined by t-test or one-way ANOVA, as appropriate.

Article Snippet: Cells were stained with anti-CD4 (RM4-5; Biolegend), fixed, permeabilized with Phosflow Perm Buffer III (BD Pharmingen) and stained with an antibody against phospho-STAT1-Y701 (p-STAT1) (58D6, Cell Signaling).

Techniques: Incubation, Control, Generated, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Lithium Controls Central Nervous System Autoimmunity through Modulation of IFN-γ Signaling

doi: 10.1371/journal.pone.0052658

Figure Lengend Snippet: EAE was induced in (A) WT (B), Stat1 −/− , (C) Ifngr1 −/− , and (D) Ifnar1 −/− as described in . Arrows indicate first day of administration of lithium. (Mean ± SEM, n = 10–13 mice/group * p <0.05, or NS, not significant, from start of treatment until day 30, as determined by Mann-Whitney test). (E) RNA was isolated from spinal cords of WT and Ifngr1 −/− mice on day 20 post-immunization and evaluated for gene expression by real-time PCR, as detailed in . n = 3 for immunized, n = 1 for unimmunized controls. * p <0.05, as determined by one-way ANOVA.

Article Snippet: Cells were stained with anti-CD4 (RM4-5; Biolegend), fixed, permeabilized with Phosflow Perm Buffer III (BD Pharmingen) and stained with an antibody against phospho-STAT1-Y701 (p-STAT1) (58D6, Cell Signaling).

Techniques: MANN-WHITNEY, Isolation, Gene Expression, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Lithium Controls Central Nervous System Autoimmunity through Modulation of IFN-γ Signaling

doi: 10.1371/journal.pone.0052658

Figure Lengend Snippet: Analysis of disease parameters for active EAE induced in untreated and lithium-treated WT, Stat1 −/− , Ifngr1 −/− and Ifnar1 −/− mice.

Article Snippet: Cells were stained with anti-CD4 (RM4-5; Biolegend), fixed, permeabilized with Phosflow Perm Buffer III (BD Pharmingen) and stained with an antibody against phospho-STAT1-Y701 (p-STAT1) (58D6, Cell Signaling).

Techniques:

Journal: Scientific Reports

Article Title: Nucleosomal dsDNA Stimulates APOL1 Expression in Human Cultured Podocytes by Activating the cGAS/IFI16-STING Signaling Pathway

doi: 10.1038/s41598-019-51998-w

Figure Lengend Snippet: nsDNA-induced APOL1 expression is partly attenuated by JAK1/JAK2 inhibitor Ruxolitinib. ( a ) Ruxolitinib (Ruxo) inhibited IFNβ-mediated phosphorylation of STAT1. Sets of AB8/13 podocytes were treated with DMSO (solvent) only (Control), treated for 2 h with 5 μM Ruxo, treated for 15 min with 10 ng ml −1 IFNβ, or pretreated with Ruxo for 2 h followed by IFNβ stimulation for 15 min (Ruxo/IFNβ). The blot images were obtained from different gels. The blot probed for P-STAT1 was re-probed for GAPDH. ( b , c ) AB8/13 podocytes were treated with Ruxo and IFNβ as indicated and subsequently transfected with 1 μg ml −1 nsDNA for 2 h ( b ) or 18 h ( c ). The IFI16/GAPDH and APOL1/GAPDH ratios in unstimulated cells (Control) were both set as 1.0. The blot images in ( b ) were obtained from different gels. The blot probed for P-IRF3 was re-probed for P-STING. Other blot images were cropped from individually probed blots. The blot images in ( c ) were obtained from different gels. The blot probed for IFI16 was re-probed for cGAS. Other blot images were cropped from individually probed blots. Full images of all blots are shown in Supplementary Fig. . ( d , e ) Ruxo abolished expression of APOL1 ( d ) and IFI16 mRNA ( e ) induced by exogenous IFNβ. ( f , g ) Ruxo partially inhibited nsDNA-induced APOL1 mRNA expression ( f ) but abolished nsDNA-induced IFI16 mRNA expression ( g ). Expression of APOL1 and IFNβ mRNA was analyzed by qRT-PCR 18 h after transfection with 1 μg ml −1 nsDNA. mRNA expression was normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).

Article Snippet: The following antibodies were used: APOL1 (Sigma, HPA018885, 1:5000), cGAS (Cell Signaling, D1D3G, 1:1000), IFI16 (Santa Cruz, sc-8023, 1:200), STING (Cell Signaling, D2P2F, 1:1000), P-STING (S366, Cell Signaling, D7C3S, 1:1000), TBK1 (Cell Signaling, D1B4, 1:500), P-TBK1 (S172, Cell Signaling, D52C2, 1:500), IRF3 (Cell Signaling, D6I4C, 1:1000), P-IRF3 (S386, Sigma, ABE501, 1:1000), STAT1 (Santa Cruz, sc-464, 1:500), P-STAT1 (Y701, Santa Cruz, sc-136229, 1:500), and GAPDH (Santa Cruz, sc-25778, 1:15000).

Techniques: Expressing, Phospho-proteomics, Solvent, Control, Transfection, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Nucleosomal dsDNA Stimulates APOL1 Expression in Human Cultured Podocytes by Activating the cGAS/IFI16-STING Signaling Pathway

doi: 10.1038/s41598-019-51998-w

Figure Lengend Snippet: Our proposed model of nsDNA-induced APOL1 expression through engagement of the cGAS/IFI16-STING pathway in human immortalized AB8/13 podocytes. Binding of cytosolic nsDNA by cGAS and IFI16 activates STING, which subsequently activates TBK1. Activated TBK1 phosphorylates IRF3, which promotes transcription of APOL1 and IFNβ . IFNβ released from the cells (or exogenous IFNβ) binds to IFNAR. IFNAR-associated JAK1 and Tyk2 kinases then phosphorylate STAT1, which promotes transcription of APOL1 and IFI16 . A putative IFI16-mediated activation of STING is indicated by a dashed arrow. A potential cooperation between cGAS and IFI16 is indicated by a double-headed arrow. Deficient STING phosphorylation observed in cGAS- or IFI16-knockdown cells (Fig. ) suggests that nsDNA-induced APOL1 expression may be mediated by a phospho-STING-independent pathway, marked by a green arrow. A dual JAK1/JAK2 inhibitor Ruxolitinib (Ruxo) suppresses STAT1 activation and thereby inhibits IFI16 expression and STAT1-mediated APOL1 expression. Since IFNAR-mediated signaling involves JAK1 but not JAK2, our model only depicts the inhibition of JAK1 by Ruxo. Ruxo does not affect IRF3-mediated APOL1 expression.

Article Snippet: The following antibodies were used: APOL1 (Sigma, HPA018885, 1:5000), cGAS (Cell Signaling, D1D3G, 1:1000), IFI16 (Santa Cruz, sc-8023, 1:200), STING (Cell Signaling, D2P2F, 1:1000), P-STING (S366, Cell Signaling, D7C3S, 1:1000), TBK1 (Cell Signaling, D1B4, 1:500), P-TBK1 (S172, Cell Signaling, D52C2, 1:500), IRF3 (Cell Signaling, D6I4C, 1:1000), P-IRF3 (S386, Sigma, ABE501, 1:1000), STAT1 (Santa Cruz, sc-464, 1:500), P-STAT1 (Y701, Santa Cruz, sc-136229, 1:500), and GAPDH (Santa Cruz, sc-25778, 1:15000).

Techniques: Expressing, Binding Assay, Activation Assay, Phospho-proteomics, Knockdown, Inhibition

Journal: PLoS Pathogens

Article Title: Out-of-Sequence Signal 3 as a Mechanism for Virus-Induced Immune Suppression of CD8 T Cell Responses

doi: 10.1371/journal.ppat.1004357

Figure Lengend Snippet: (A–H) Mice were HBSS- (open bars) or poly(I∶C)- (black bars) treated once 1, 2, or 3 days prior to isolation. (A–D) Splenocytes were isolated, stimulated ex vivo with IFNβ for 30 min and then stained for phosphoSTATs. Cells were gated on CD44loCD8α+ T cells plotting pSTAT MFI after IFNβ stimulation showing MFI for (A) pSTAT1, (B) pSTAT3 (C) pSTAT4 or (D) pSTAT5. (E–H) Splenocytes were stained ex vivo for total STAT proteins including (E) STAT1, (F) STAT3, (G) STAT4, and (H) STAT5. Plots showing total STAT MFI, gated on CD44loCD8α+T cells. Data are representative of 2 independent experiments with n of 3 mice per group.

Article Snippet: Splenocytes were washed and then stained with a combination of fluorescently labeled Abs pY701 STAT1 (BD Bioscience), pY705 STAT3 (BD Bioscience), pY693 STAT4 (BD Bioscience), pY694 STAT5 (BD Bioscience), STAT1 (clone 1/Stat1; BD Bioscience), or unlabeled Abs STAT3 (79D7; cell signaling technology), STAT4 (C46B10; cell signaling technology), STAT5 (3H7; cell signaling technology), SOCS1 (A156; cell signaling technology), or BCL3 (C-14; Santa Cruz Biotechnology) for 20–30 min at RT in the dark.

Techniques: Isolation, Ex Vivo, Staining

Journal: Journal of Biological Chemistry

Article Title: Type I Interferon Signaling Is Decoupled from Specific Receptor Orientation through Lenient Requirements of the Transmembrane Domain

doi: 10.1074/jbc.m115.686071

Figure Lengend Snippet: FIGURE 1. Inserting one to five alanine residues near the N terminus of the TMD of IFNAR1 has little effect on binding and activity. A, ribbon represen- tation of the ternary complex of IFNAR1, IFNAR2, and IFN (based on Protein Data Bank 3SE3) (7) and the location of the additional alanine residues inserted within the transmembrane domain of IFNAR1. To the left is a schematic representation of the expected effect of adding one to five alanine residues (A1–A5). B, in situ binding curves of the different IFNAR1 mutants. Signal emitted from 125I-labeled wild-type IFN2 was measured after competing with cold IFN-YNS at different concentrations. The y axis represents the fraction of signal relative to the signal in the absence of cold competitor. As control, we also measured binding to non-transfected cells and to cells transfected with the IFNAR1 mutant L163C. IC50 values were calculated by fitting the normalized data using KaleidaGraph 4.1. C, A1–A5 mutant IFNAR1 HUH7 cells were treated for 30 min with 1 nM IFN2 and then analyzed by Western blotting using antibodies for pSTAT1 and pSTAT2. After stripping, the blots were re-analyzed with anti-STAT1 and -STAT2 antibodies. The graph on the right shows the normalized (to total and untreated) levels of phosphorylation. D, gene expressions of transiently transfected HUH7 cells after 16 h of treatment with 0.5–1000 pM IFN2. qPCR was then performed for IFI6 and MX1 genes as indicated. The data presented are the relative expression levels compared with those of untreated cells, normalized against HPRT1. The results are average and standard error of three independent experiments. E, fold change in gene expression using the Fluidigm system (see “Experimental Procedures”). Cells were treated as in D, and cDNAs (50 ng/ml) were preamplified with all the primers polled and analyzed with the BioMark real time PCR instrument. Data are presented using the NetWalker analysis tool. The upper eight genes are tunable genes, and the lower 12 genes are robust genes (15). The colors represent the value of a given gene (rows) in a specific mutant and concentration of interferon (columns). Genes with a high Ct value (high expression)areinred,andgeneswithalowCtvalue(lowexpression)areinblue.Comparisonsofthefoldchangesweredonerelativetothoseofuntreated cells. Data are representative of two independent experiments.

Article Snippet: Monoclonal antiSTAT1 and anti-pY701-STAT1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Activity Assay, In Situ, Labeling, Control, Transfection, Mutagenesis, Western Blot, Stripping Membranes, Phospho-proteomics, Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Type I Interferon Signaling Is Decoupled from Specific Receptor Orientation through Lenient Requirements of the Transmembrane Domain

doi: 10.1074/jbc.m115.686071

Figure Lengend Snippet: FIGURE 3. Inserting one to four alanine residues near the C terminus of the TMD of IFNAR1 (T1 to T4) has little effect on binding and activity. A, the locationoftheinsertedalaninemutations.B,insitubindingcurvesofthedifferentIFNAR1mutants.C,analyzingpSTAT1,pSTAT2,STAT1,andSTAT2levelsupon 1 nM IFN2 induction for 30 min using specific antibodies. D, gene expressions for IFI6 and MX1 upon IFN induction at the given concentrations. E, fold change in gene expression for a set of tunable and robust genes upon IFN induction. Further experimental details are provided in the legend of Fig. 1.

Article Snippet: Monoclonal antiSTAT1 and anti-pY701-STAT1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Activity Assay, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Type I Interferon Signaling Is Decoupled from Specific Receptor Orientation through Lenient Requirements of the Transmembrane Domain

doi: 10.1074/jbc.m115.686071

Figure Lengend Snippet: FIGURE 4. Mutating the immediate surrounding of the TMD of IFNAR1. A, left panel shows insertions of 2, 4, and 10 serine residues between the TMD and the extracellular domain. In addition, the two C-terminal residues of the TMD, Lys-458 and Val-459 were mutated to alanine. Finally, the TMD of IFNAR1 was replaced with that of IFNAR2 (making a homodimeric TMD, right panel). B, in situ binding curves of the different IFNAR1 mutants. Binding of the S10 mutant was 5-fold weaker than wild type and the other mutants analyzed. C, pSTAT1, pSTAT2, STAT1, and STAT2 levels upon 1 nM IFN2 induction for 30 min using specific antibodies. D, gene expressions for IFI6 and MX1 upon IFN induction at the given concentrations. One asterisk is for a p value of 0.05, and two asterisks is for p values of 0.01 (by one-way analysis of variance). E, fold change in gene expression for a set of tunable and robust genes upon IFN induction. Further experimental details are provided in the legend of Fig. 1.

Article Snippet: Monoclonal antiSTAT1 and anti-pY701-STAT1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Situ, Binding Assay, Mutagenesis, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Type I Interferon Signaling Is Decoupled from Specific Receptor Orientation through Lenient Requirements of the Transmembrane Domain

doi: 10.1074/jbc.m115.686071

Figure Lengend Snippet: FIGURE 6. STAT phosphorylation and gene activation measured at different times of IFN induction. A, WT, S10, TMR1R2, and T3 IFNAR1 constructs were transfected into HUH7 cells, which were treated for 60 min with 1 nM IFN2 and then analyzed by Western blotting using antibodies for pSTAT1 and pSTAT2. After stripping, the blots were re-analyzed with anti-STAT1 and -STAT2 antibodies. On the right is a comparison with 30 min of IFN activation of WT IFNAR1. B, gene expressions of transiently transfected HUH7 cells with WT, A1, T3, TMR1R2, and S10 IFNAR1 after 8 and 24 h treatment with 0.5 and 10 pM IFN2. qPCR was then performed for IFI6 and MX1 genes as indicated. The data presented are the relative expression levels compared with those of untreated cells, normalized against HPRT1.

Article Snippet: Monoclonal antiSTAT1 and anti-pY701-STAT1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Phospho-proteomics, Activation Assay, Construct, Transfection, Western Blot, Stripping Membranes, Comparison, Expressing