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    • rabbit anti irf3  (Cell Signaling Technology Inc)
    98
    Cell Signaling Technology Inc rabbit anti irf3
    Rabbit Anti Irf3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti irf3/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti irf3 - by Bioz Stars, 2023-03
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    rb a irf3 s396  (Cell Signaling Technology Inc)
    96
    Cell Signaling Technology Inc rb a irf3 s396
    Rb A Irf3 S396, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rb a irf3 s396/product/Cell Signaling Technology Inc
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rb a irf3 s396 - by Bioz Stars, 2023-03
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    irf3 xp antibody  (Cell Signaling Technology Inc)
    96
    Cell Signaling Technology Inc irf3 xp antibody
    Graphical abstract. a) Brief schematic of anti-viral signaling pathways. Viral DNA binds to cGAS, which produces 2′3′-cGAMP. 2′3′-cGAMP binds to STING which activates the transcription factor <t>IRF3</t> via the kinase Tbk1. NFkB is also activated, likely by the IKK kinases, though this effect has not been as thoroughly studied. Other anti-viral pathways also activate IRF3 and NFkB, by similar mechanism. The transcription factors promote the expression of cytokines, which ultimately get secreted. b) Schematic of the screening approach. In the primary screen, small molecules hits were identified in primary macrophages by an IRF3/NFkB nuclear translocation screen. Secondary screens focused on whether these hits modulated gene expression at later time points, and subsequent work focused on identifying the mechanism of action of these hits
    Irf3 Xp Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irf3 xp antibody/product/Cell Signaling Technology Inc
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    irf3 xp antibody - by Bioz Stars, 2023-03
    96/100 stars
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    anti phospho irf3  (Cell Signaling Technology Inc)
    96
    Cell Signaling Technology Inc anti phospho irf3
    Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase <t>IRF3</t> activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P < 0.05; ***, P < 0.005. n.s., no significant difference.
    Anti Phospho Irf3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 1 article reviews
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    anti phospho irf3 - by Bioz Stars, 2023-03
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    mouse phospho s379 irf3  (Cell Signaling Technology Inc)
    92
    Cell Signaling Technology Inc mouse phospho s379 irf3
    M04 induces canonical activation of <t>IRF3</t> which is essential to reporter signal generated by the compound. (A) Immunoblot showing phosphorylation status of TBK1 Ser172 and IRF3 Ser386 as well as corresponding total protein levels in MM6 cells (left) and THF (right) exposed for 4 h to 1% DMSO, 50 μM M04, or 1,000 HAU/mL SeV as indicated; (B) Indirect immunofluorescence showing subcellular localization of IRF3 in THF exposed for 4 h to 1% DMSO, transfected 2'3'cGAMP (10 μg/mL), 100 ng/mL TNFα, or 50 μM M04; (C) Reporter assay illustrating IFN-dependent LUC induction following overnight treatment with 1% DMSO, 1,000 U/mL IFNβ, 1,000 HAU/mL SeV, or 50 μM M04 in parental cells as well as those from which IRF3 was deleted as indicated. Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells based on quadruplicate measurements. Student's T -test was used to compare RLU in the parental and ΔIRF3 cells ** P < 0.01; *** P < 0.001.
    Mouse Phospho S379 Irf3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse phospho s379 irf3/product/Cell Signaling Technology Inc
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse phospho s379 irf3 - by Bioz Stars, 2023-03
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    Image Search Results


    Graphical abstract. a) Brief schematic of anti-viral signaling pathways. Viral DNA binds to cGAS, which produces 2′3′-cGAMP. 2′3′-cGAMP binds to STING which activates the transcription factor IRF3 via the kinase Tbk1. NFkB is also activated, likely by the IKK kinases, though this effect has not been as thoroughly studied. Other anti-viral pathways also activate IRF3 and NFkB, by similar mechanism. The transcription factors promote the expression of cytokines, which ultimately get secreted. b) Schematic of the screening approach. In the primary screen, small molecules hits were identified in primary macrophages by an IRF3/NFkB nuclear translocation screen. Secondary screens focused on whether these hits modulated gene expression at later time points, and subsequent work focused on identifying the mechanism of action of these hits

    Journal: ACS chemical biology

    Article Title: A high content screen in macrophages identifies small molecule modulators of STING-IRF3 and NFkB signaling

    doi: 10.1021/acschembio.7b01060

    Figure Lengend Snippet: Graphical abstract. a) Brief schematic of anti-viral signaling pathways. Viral DNA binds to cGAS, which produces 2′3′-cGAMP. 2′3′-cGAMP binds to STING which activates the transcription factor IRF3 via the kinase Tbk1. NFkB is also activated, likely by the IKK kinases, though this effect has not been as thoroughly studied. Other anti-viral pathways also activate IRF3 and NFkB, by similar mechanism. The transcription factors promote the expression of cytokines, which ultimately get secreted. b) Schematic of the screening approach. In the primary screen, small molecules hits were identified in primary macrophages by an IRF3/NFkB nuclear translocation screen. Secondary screens focused on whether these hits modulated gene expression at later time points, and subsequent work focused on identifying the mechanism of action of these hits

    Article Snippet: IRF3 XP antibody (Cell Signaling, 11904 T) and NFkB antibody (Santa Cruz, sc-8008) were adjusted to final dilution of 1:400.

    Techniques: Expressing, Translocation Assay

    Summary of activator screen. a) Representative images from control group wells. cGAMP activates IRF3, CL075 activates NFkB, and LPS activates both. b) Quantification of IRF3 and NFkB nuclear fractions for control groups (left). Replicate analysis for IRF3 scores (middle) and for NFkB scores (right) c) Waterfall plot for IRF3 and NFkB activators. Scores for each compound were computed by taking the maximum effect achieved out of the doses considered. The hit rates were low, especially for IRF3 activators.

    Journal: ACS chemical biology

    Article Title: A high content screen in macrophages identifies small molecule modulators of STING-IRF3 and NFkB signaling

    doi: 10.1021/acschembio.7b01060

    Figure Lengend Snippet: Summary of activator screen. a) Representative images from control group wells. cGAMP activates IRF3, CL075 activates NFkB, and LPS activates both. b) Quantification of IRF3 and NFkB nuclear fractions for control groups (left). Replicate analysis for IRF3 scores (middle) and for NFkB scores (right) c) Waterfall plot for IRF3 and NFkB activators. Scores for each compound were computed by taking the maximum effect achieved out of the doses considered. The hit rates were low, especially for IRF3 activators.

    Article Snippet: IRF3 XP antibody (Cell Signaling, 11904 T) and NFkB antibody (Santa Cruz, sc-8008) were adjusted to final dilution of 1:400.

    Techniques:

    Distributions of IRF3 and NFkB nuclear fractions for 5 cGAMP treated wells (blue) and H2O treated wells (red). Each line represents the distribution in a single well.

    Journal: ACS chemical biology

    Article Title: A high content screen in macrophages identifies small molecule modulators of STING-IRF3 and NFkB signaling

    doi: 10.1021/acschembio.7b01060

    Figure Lengend Snippet: Distributions of IRF3 and NFkB nuclear fractions for 5 cGAMP treated wells (blue) and H2O treated wells (red). Each line represents the distribution in a single well.

    Article Snippet: IRF3 XP antibody (Cell Signaling, 11904 T) and NFkB antibody (Santa Cruz, sc-8008) were adjusted to final dilution of 1:400.

    Techniques:

    List of selected agonists. Compounds 1–30 were selected as cGAMP like at 10 uM or less. Compounds 31–36 were selected IRF3 inducers from the preliminary analysis

    Journal: ACS chemical biology

    Article Title: A high content screen in macrophages identifies small molecule modulators of STING-IRF3 and NFkB signaling

    doi: 10.1021/acschembio.7b01060

    Figure Lengend Snippet: List of selected agonists. Compounds 1–30 were selected as cGAMP like at 10 uM or less. Compounds 31–36 were selected IRF3 inducers from the preliminary analysis

    Article Snippet: IRF3 XP antibody (Cell Signaling, 11904 T) and NFkB antibody (Santa Cruz, sc-8008) were adjusted to final dilution of 1:400.

    Techniques:

    Summary of the inhibitor screen. a) Sample images from control groups. The Tbk1 inhibitors, BX795 and MRT67307 block IRF3 nuclear fraction b) Quantification of control group IRF3 fraction (left) and replicate analysis (right) c) Waterfall plot for the inhibitor screen. Score for each compound was computed by taking the maximum effect. Both inhibitors and potentiators can be resolved. d) Summary of chemical class for inhibitors shown in table 2. Kinase inhibitors and natural product antibiotic are enriched.

    Journal: ACS chemical biology

    Article Title: A high content screen in macrophages identifies small molecule modulators of STING-IRF3 and NFkB signaling

    doi: 10.1021/acschembio.7b01060

    Figure Lengend Snippet: Summary of the inhibitor screen. a) Sample images from control groups. The Tbk1 inhibitors, BX795 and MRT67307 block IRF3 nuclear fraction b) Quantification of control group IRF3 fraction (left) and replicate analysis (right) c) Waterfall plot for the inhibitor screen. Score for each compound was computed by taking the maximum effect. Both inhibitors and potentiators can be resolved. d) Summary of chemical class for inhibitors shown in table 2. Kinase inhibitors and natural product antibiotic are enriched.

    Article Snippet: IRF3 XP antibody (Cell Signaling, 11904 T) and NFkB antibody (Santa Cruz, sc-8008) were adjusted to final dilution of 1:400.

    Techniques: Blocking Assay

    Re-test of MAPKAPK5 inhibitors. a) Enrichment strategy to identify relevant kinase targets in inhibitor list. b) Structures of MAPKAPK5 inhibitors. INHIB1 and INHIB2 are active, while INHIB3 has much weaker affinity for MAPKAPK5. c) Re-test of INHIBs 1,2,3 in imaging assay in a single donor. Shown is the IRF3 distribution in cells treated with inhibitor (10 uM, 2 hour pre-treatment) and 2′3′ cGAMP (75 uM, 2 hour stimulation). d) Western blot measuring IRF3 phosphorylation (Ser396) after 4 hour stimulation with 2′3′ cGAMP (62.5 uM) in the presence or absence of inhibitor (10 uM). Cells were pre-treated with inhibitor for about 2 hours. Shown below is quantification of pIRF3 levels normalized to tubulin. e) Mini-SAR of 9 tetracycle (INHIB2-like) MAPKAPK5 inhibitors. IP-10 levels are measured (left), and biological activity is plotted against MAPKAPK5 IC50 (right).

    Journal: ACS chemical biology

    Article Title: A high content screen in macrophages identifies small molecule modulators of STING-IRF3 and NFkB signaling

    doi: 10.1021/acschembio.7b01060

    Figure Lengend Snippet: Re-test of MAPKAPK5 inhibitors. a) Enrichment strategy to identify relevant kinase targets in inhibitor list. b) Structures of MAPKAPK5 inhibitors. INHIB1 and INHIB2 are active, while INHIB3 has much weaker affinity for MAPKAPK5. c) Re-test of INHIBs 1,2,3 in imaging assay in a single donor. Shown is the IRF3 distribution in cells treated with inhibitor (10 uM, 2 hour pre-treatment) and 2′3′ cGAMP (75 uM, 2 hour stimulation). d) Western blot measuring IRF3 phosphorylation (Ser396) after 4 hour stimulation with 2′3′ cGAMP (62.5 uM) in the presence or absence of inhibitor (10 uM). Cells were pre-treated with inhibitor for about 2 hours. Shown below is quantification of pIRF3 levels normalized to tubulin. e) Mini-SAR of 9 tetracycle (INHIB2-like) MAPKAPK5 inhibitors. IP-10 levels are measured (left), and biological activity is plotted against MAPKAPK5 IC50 (right).

    Article Snippet: IRF3 XP antibody (Cell Signaling, 11904 T) and NFkB antibody (Santa Cruz, sc-8008) were adjusted to final dilution of 1:400.

    Techniques: Imaging, Western Blot, Activity Assay

    Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P < 0.05; ***, P < 0.005. n.s., no significant difference.

    Journal: Journal of Virology

    Article Title: Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation

    doi: 10.1128/JVI.02843-15

    Figure Lengend Snippet: Mutations at the second and third in-frame AUG codons in NS1 ORF of PR8 virus increase IRF3 activation and IFN-β production in infected cells. A549 cells were infected with WT or mutated influenza A virus that expresses NS1 with mutations at the second and third AUG (codons 79 and 81, respectively) in the PR8 backbone (A and B) or Ud backbone (E and F) at an MOI of 2. (A and E) At 9 h postinfection, cell extracts were collected and analyzed by immunoblotting with anti-NS1, anti-phospho-IRF3 (serine 396), anti-total IRF3, and anti-β actin antibodies. (B and F) Relative amount of IFN-β mRNA of infected cells was quantitated by real-time RT-PCR. HEL-299 cells were infected with the WT or the PR8 virus mutated at codons 79 and 81 at an MOI of 2. (C) At 9 h postinfection, cell extracts were collected and analyzed for the proteins of NS1, phosphorylated IRF3, and total IRF3. (D) Relative amount of IFN-β pre-mRNA and mRNA of infected cells was quantitated by real-time RT-PCR. tNS1, truncated NS1 protein. *, P < 0.05; ***, P < 0.005. n.s., no significant difference.

    Article Snippet: Other antibodies were anti-total IRF3 (Santa Cruz Biotechnology, USA), anti-phospho-IRF3 (Ser396; Cell Signaling, USA), anti-β-actin (Sigma-Aldrich, USA), and horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare, USA).

    Techniques: Activation Assay, Infection, Western Blot, Quantitative RT-PCR

    N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

    Journal: Journal of Virology

    Article Title: Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation

    doi: 10.1128/JVI.02843-15

    Figure Lengend Snippet: N-terminally truncated NS1 protein of PR8 virus inhibits IFN-β promoter activation. (A) 293T cells were cotransfected with a reporter plasmid containing IFN-β promoter fused to the firefly luciferase gene, a renilla luciferase control plasmid, a plasmid expressing WT or truncated NS1 protein (containing amino acids 79 to 230 derived from PR8 virus), and either the plasmid expressing Myc-tagged RIG-I CARD domains or an empty vector. At 24 h posttransfection, cell extracts were collected and analyzed by dual-luciferase activity assay. (B) 293T cells were cotransfected with the aforementioned luciferase reporters and the plasmid expressing PR8 WT or truncated NS1 for 18 h, followed by Sendai virus (SeV) infection for another 24 h. IFN-β promoter activity was determined by dual-luciferase activity assay. A549 cells that constitutionally express RFP or PR8 truncated NS1 were infected with Sendai virus at a final concentration of 13 HAU/ml (C) or with PR/M79.81I (D) or Ud/M79.81I (E) mutant virus at an MOI of 0.2. At 9 h postinfection, the activation of IRF3 was examined by anti-phosphorylated IRF3 antibody. The relative levels of IFN-β mRNA or pre-mRNA in the infected cells were analyzed by real-time RT-PCR. Protein expression was monitored by immunoblotting with anti-NS1, β-actin, c-Myc, or total IRF3 antibodies. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

    Article Snippet: Other antibodies were anti-total IRF3 (Santa Cruz Biotechnology, USA), anti-phospho-IRF3 (Ser396; Cell Signaling, USA), anti-β-actin (Sigma-Aldrich, USA), and horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare, USA).

    Techniques: Activation Assay, Plasmid Preparation, Luciferase, Expressing, Derivative Assay, Activity Assay, Infection, Concentration Assay, Mutagenesis, Quantitative RT-PCR, Western Blot

    M04 induces canonical activation of IRF3 which is essential to reporter signal generated by the compound. (A) Immunoblot showing phosphorylation status of TBK1 Ser172 and IRF3 Ser386 as well as corresponding total protein levels in MM6 cells (left) and THF (right) exposed for 4 h to 1% DMSO, 50 μM M04, or 1,000 HAU/mL SeV as indicated; (B) Indirect immunofluorescence showing subcellular localization of IRF3 in THF exposed for 4 h to 1% DMSO, transfected 2'3'cGAMP (10 μg/mL), 100 ng/mL TNFα, or 50 μM M04; (C) Reporter assay illustrating IFN-dependent LUC induction following overnight treatment with 1% DMSO, 1,000 U/mL IFNβ, 1,000 HAU/mL SeV, or 50 μM M04 in parental cells as well as those from which IRF3 was deleted as indicated. Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells based on quadruplicate measurements. Student's T -test was used to compare RLU in the parental and ΔIRF3 cells ** P < 0.01; *** P < 0.001.

    Journal: Frontiers in Immunology

    Article Title: Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner

    doi: 10.3389/fimmu.2020.01430

    Figure Lengend Snippet: M04 induces canonical activation of IRF3 which is essential to reporter signal generated by the compound. (A) Immunoblot showing phosphorylation status of TBK1 Ser172 and IRF3 Ser386 as well as corresponding total protein levels in MM6 cells (left) and THF (right) exposed for 4 h to 1% DMSO, 50 μM M04, or 1,000 HAU/mL SeV as indicated; (B) Indirect immunofluorescence showing subcellular localization of IRF3 in THF exposed for 4 h to 1% DMSO, transfected 2'3'cGAMP (10 μg/mL), 100 ng/mL TNFα, or 50 μM M04; (C) Reporter assay illustrating IFN-dependent LUC induction following overnight treatment with 1% DMSO, 1,000 U/mL IFNβ, 1,000 HAU/mL SeV, or 50 μM M04 in parental cells as well as those from which IRF3 was deleted as indicated. Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells based on quadruplicate measurements. Student's T -test was used to compare RLU in the parental and ΔIRF3 cells ** P < 0.01; *** P < 0.001.

    Article Snippet: Sources and concentrations of antibodies used against the following antigens are indicated in parentheses: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SC-51906; Santa Cruz) (1:10,000), IRF3 (4302; Cell Signaling), human phospho-IRF3 (76493; Abcam), mouse IRF3 (SC-9082; Santa Cruz), mouse phospho-S379 IRF3 (79945S; Cell Signaling), mouse phospho-S396 IRF3 (29047S; Cell Signaling), STING (13647S; Cell Signaling), phospho-S366 STING (19781S; Cell Signaling), TBK1 (3504S; Cell Signaling), phospho-TBK1 (5483S; Cell Signaling), NF-kB P65 (SC372; Santa Cruz), NF-kB P50 (3035; Cell Signaling), GM-130 (610823; BD Biosciences).

    Techniques: Activation Assay, Generated, Western Blot, Immunofluorescence, Transfection, Reporter Assay

    Innate Activation by M04 requires STING but not MAVS, TRIF, or cytosolic DNA PRRs. (A) Reporter assay illustrating IFN-dependent LUC induction in THF-ISRE-ΔMAVS/TRIF following overnight treatment with 1% DMSO, transfected cGAMP (10 μg/mL), or 75 μM M04. Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells as the basis ( n = 4 treatments); (B) Immunoblot showing phosphorylation status of IRF3 Ser386, total IRF3, and GAPDH in THF-ISRE-ΔMAVS/TRIF following 8 h treatment with 1% DMSO, 75 μM M04, 1,000 HAU/mL SeV or 25 μM ABZI as indicated; (C) Reporter assay illustrating IFN-dependent LUC induction in THF-ISRE-ΔSTING following overnight treatment with 1% DMSO, 1,000 U/mL IFNβ, 1,000 HAU/mL SeV, or 75 μM M04. Data presented are mean RLU ± SD as described above; Student's T -test was used to compare RLU *** p < 0.001; (D) Immunoblot showing phosphorylation status of IRF3 Ser386, total IRF3 in THF-ISRE-ΔSTING following 4 h treatment with 1% DMSO, 50 μM M04, 1,000 HAU/mL SeV or 25 μM ABZI as indicated; (E) Secretion of bioactive type I IFN from parental THF as well as THF-ISRE-ΔMAVS/TRIF and THF-ISRE-ΔSTING treated in triplicate overnight with 1% DMSO, 1,000 HAU/mL SeV, transfected cGAMP (10 μg/mL), or 75 μM M04. Data are expressed as mean concentrations ± SD for IFNβ equivalent units. Statistical significance between treated and untreated cells of similar genetic background was calculated using Student's T -test. **** p < 0.0001; (F) Reporter assay from WT parental THF-ISRE cells as well as from cells from which indicated dsDNA-specific PRRs were deleted. Values presented are mean fold changes ± SD for duplicates relative to the value for DMSO-treated cells.

    Journal: Frontiers in Immunology

    Article Title: Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner

    doi: 10.3389/fimmu.2020.01430

    Figure Lengend Snippet: Innate Activation by M04 requires STING but not MAVS, TRIF, or cytosolic DNA PRRs. (A) Reporter assay illustrating IFN-dependent LUC induction in THF-ISRE-ΔMAVS/TRIF following overnight treatment with 1% DMSO, transfected cGAMP (10 μg/mL), or 75 μM M04. Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells as the basis ( n = 4 treatments); (B) Immunoblot showing phosphorylation status of IRF3 Ser386, total IRF3, and GAPDH in THF-ISRE-ΔMAVS/TRIF following 8 h treatment with 1% DMSO, 75 μM M04, 1,000 HAU/mL SeV or 25 μM ABZI as indicated; (C) Reporter assay illustrating IFN-dependent LUC induction in THF-ISRE-ΔSTING following overnight treatment with 1% DMSO, 1,000 U/mL IFNβ, 1,000 HAU/mL SeV, or 75 μM M04. Data presented are mean RLU ± SD as described above; Student's T -test was used to compare RLU *** p < 0.001; (D) Immunoblot showing phosphorylation status of IRF3 Ser386, total IRF3 in THF-ISRE-ΔSTING following 4 h treatment with 1% DMSO, 50 μM M04, 1,000 HAU/mL SeV or 25 μM ABZI as indicated; (E) Secretion of bioactive type I IFN from parental THF as well as THF-ISRE-ΔMAVS/TRIF and THF-ISRE-ΔSTING treated in triplicate overnight with 1% DMSO, 1,000 HAU/mL SeV, transfected cGAMP (10 μg/mL), or 75 μM M04. Data are expressed as mean concentrations ± SD for IFNβ equivalent units. Statistical significance between treated and untreated cells of similar genetic background was calculated using Student's T -test. **** p < 0.0001; (F) Reporter assay from WT parental THF-ISRE cells as well as from cells from which indicated dsDNA-specific PRRs were deleted. Values presented are mean fold changes ± SD for duplicates relative to the value for DMSO-treated cells.

    Article Snippet: Sources and concentrations of antibodies used against the following antigens are indicated in parentheses: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SC-51906; Santa Cruz) (1:10,000), IRF3 (4302; Cell Signaling), human phospho-IRF3 (76493; Abcam), mouse IRF3 (SC-9082; Santa Cruz), mouse phospho-S379 IRF3 (79945S; Cell Signaling), mouse phospho-S396 IRF3 (29047S; Cell Signaling), STING (13647S; Cell Signaling), phospho-S366 STING (19781S; Cell Signaling), TBK1 (3504S; Cell Signaling), phospho-TBK1 (5483S; Cell Signaling), NF-kB P65 (SC372; Santa Cruz), NF-kB P50 (3035; Cell Signaling), GM-130 (610823; BD Biosciences).

    Techniques: Activation Assay, Reporter Assay, Transfection, Western Blot

    Responsiveness to M04 can be Conferred through Introduction of WT STING Allelic Variant. (A) Reporter assay illustrating IFN-dependent LUC induction in THP-1-ISG-Lucia following overnight treatment with 1% DMSO, 1,000 HAU/mL SeV, 1,000 U/mL IFNβ, 75 μM TRIF agonist AV-C, 25 μM ABZI, or 75 μM M04. Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells as the basis ( n = 4 treatments). Student's T -test was used to compare RLU *** p < 0.001, **** p < 0.0001; (B) lmmunoblot showing phosphorylation status of IRF3 Ser386 and total IRF3 in THP-1 whole cell lysates following 4 h treatment with 1% DMSO, 50 μM M04, 1,000 HAU/mL SeV, 25 μM ABZI, or 10 μg/mL cGAMP as indicated; (C) lmmunoblot showing expression of endogenous or ectopically expressed WT hSTING in THP-1 as indicated. (D) Immunoblot showing phosphorylation status of IRF3 Ser386 in THP-1 cells from which endogenous STING was deleted and WT STING stably introduced following indicated treatment as described above.

    Journal: Frontiers in Immunology

    Article Title: Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner

    doi: 10.3389/fimmu.2020.01430

    Figure Lengend Snippet: Responsiveness to M04 can be Conferred through Introduction of WT STING Allelic Variant. (A) Reporter assay illustrating IFN-dependent LUC induction in THP-1-ISG-Lucia following overnight treatment with 1% DMSO, 1,000 HAU/mL SeV, 1,000 U/mL IFNβ, 75 μM TRIF agonist AV-C, 25 μM ABZI, or 75 μM M04. Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells as the basis ( n = 4 treatments). Student's T -test was used to compare RLU *** p < 0.001, **** p < 0.0001; (B) lmmunoblot showing phosphorylation status of IRF3 Ser386 and total IRF3 in THP-1 whole cell lysates following 4 h treatment with 1% DMSO, 50 μM M04, 1,000 HAU/mL SeV, 25 μM ABZI, or 10 μg/mL cGAMP as indicated; (C) lmmunoblot showing expression of endogenous or ectopically expressed WT hSTING in THP-1 as indicated. (D) Immunoblot showing phosphorylation status of IRF3 Ser386 in THP-1 cells from which endogenous STING was deleted and WT STING stably introduced following indicated treatment as described above.

    Article Snippet: Sources and concentrations of antibodies used against the following antigens are indicated in parentheses: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SC-51906; Santa Cruz) (1:10,000), IRF3 (4302; Cell Signaling), human phospho-IRF3 (76493; Abcam), mouse IRF3 (SC-9082; Santa Cruz), mouse phospho-S379 IRF3 (79945S; Cell Signaling), mouse phospho-S396 IRF3 (29047S; Cell Signaling), STING (13647S; Cell Signaling), phospho-S366 STING (19781S; Cell Signaling), TBK1 (3504S; Cell Signaling), phospho-TBK1 (5483S; Cell Signaling), NF-kB P65 (SC372; Santa Cruz), NF-kB P50 (3035; Cell Signaling), GM-130 (610823; BD Biosciences).

    Techniques: Variant Assay, Reporter Assay, Expressing, Western Blot, Stable Transfection

    Transient transfection of vectors encoding WT and R232H but not HAQ hSTING confer responsiveness to M04. (A) lmmunoblot from HEK293T whole cell lysates showing expression of indicated STING variants following transient transfection, S386 phosphorylation status of IRF3 and total IRF3. Cells were left untreated or exposed to 75 μM M04, 100 nM diABZI, or 10 μg/mL cGAMP as indicated; (B) Reporter assay using cells ( n = 4) treated as described in (A) . Values displayed are mean fold changes ± SD relative to cells transfected with empty vector; (C) Expression of IFIT1 and Viperin mRNA as determined by qPCR in parental A549 cells as well as those transduced with hSTING following treatment with 1% DMSO or 75 μM M04. Data are mean fodl changes ± SD relative to DMSO-treated cells based on duplicates; (D) Synthesis of cGAMP by A549-hSTING cells as determined by ELISA following overnight treatment with 1% DMSO, HCMV, or M04. Data presented are mean pg/mL ± SD based on duplicate samples. Student's T -test was used to compare RLU and mRNA levels *** p < 0.001, **** p < 0.0001.

    Journal: Frontiers in Immunology

    Article Title: Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner

    doi: 10.3389/fimmu.2020.01430

    Figure Lengend Snippet: Transient transfection of vectors encoding WT and R232H but not HAQ hSTING confer responsiveness to M04. (A) lmmunoblot from HEK293T whole cell lysates showing expression of indicated STING variants following transient transfection, S386 phosphorylation status of IRF3 and total IRF3. Cells were left untreated or exposed to 75 μM M04, 100 nM diABZI, or 10 μg/mL cGAMP as indicated; (B) Reporter assay using cells ( n = 4) treated as described in (A) . Values displayed are mean fold changes ± SD relative to cells transfected with empty vector; (C) Expression of IFIT1 and Viperin mRNA as determined by qPCR in parental A549 cells as well as those transduced with hSTING following treatment with 1% DMSO or 75 μM M04. Data are mean fodl changes ± SD relative to DMSO-treated cells based on duplicates; (D) Synthesis of cGAMP by A549-hSTING cells as determined by ELISA following overnight treatment with 1% DMSO, HCMV, or M04. Data presented are mean pg/mL ± SD based on duplicate samples. Student's T -test was used to compare RLU and mRNA levels *** p < 0.001, **** p < 0.0001.

    Article Snippet: Sources and concentrations of antibodies used against the following antigens are indicated in parentheses: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SC-51906; Santa Cruz) (1:10,000), IRF3 (4302; Cell Signaling), human phospho-IRF3 (76493; Abcam), mouse IRF3 (SC-9082; Santa Cruz), mouse phospho-S379 IRF3 (79945S; Cell Signaling), mouse phospho-S396 IRF3 (29047S; Cell Signaling), STING (13647S; Cell Signaling), phospho-S366 STING (19781S; Cell Signaling), TBK1 (3504S; Cell Signaling), phospho-TBK1 (5483S; Cell Signaling), NF-kB P65 (SC372; Santa Cruz), NF-kB P50 (3035; Cell Signaling), GM-130 (610823; BD Biosciences).

    Techniques: Transfection, Expressing, Reporter Assay, Plasmid Preparation, Transduction, Enzyme-linked Immunosorbent Assay

    Responsiveness to M04 can be conferred to murine cells by ectopic expression of human STING-WT variant. (A) Reporter assay illustrating IFN-dependent LUC induction in RAW264.7-ISG-Lucia cells followni g overnight treatment with 1% DMSO, 160 HAU/mL SeV, 25 μM DMXAA, or 75 μM M04.Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells as the basis ( n = 4 treatments); (B) qPCR examining in vivo ISG induction following IP injection of DMXAA or M04; (C) lmmunoblot showing expression of endogenous or ectopically expressed hSTING-WT in RAW264.7 cells as indicated; (D) lmmunoblot showing phosphorylation status of IRF3 Ser379 and Ser396 as well as total IRF3 in RAW264.7-hSTING cells following 4 h treatment with 1% DMSO, 75 μM M04, 160 HAU/mL SeV, or transfection of cGAMP as indicated; (E) qPCR examining transcription of IFIT1 or Viperin following overnight treatment of parental RAW264.7 and RAW264.7-hSTING cells with 75 μM M04 ( n = 3). Data presented are mean fold changes ± SD of mRNA relative to cells treated with 1% DMSO.

    Journal: Frontiers in Immunology

    Article Title: Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner

    doi: 10.3389/fimmu.2020.01430

    Figure Lengend Snippet: Responsiveness to M04 can be conferred to murine cells by ectopic expression of human STING-WT variant. (A) Reporter assay illustrating IFN-dependent LUC induction in RAW264.7-ISG-Lucia cells followni g overnight treatment with 1% DMSO, 160 HAU/mL SeV, 25 μM DMXAA, or 75 μM M04.Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells as the basis ( n = 4 treatments); (B) qPCR examining in vivo ISG induction following IP injection of DMXAA or M04; (C) lmmunoblot showing expression of endogenous or ectopically expressed hSTING-WT in RAW264.7 cells as indicated; (D) lmmunoblot showing phosphorylation status of IRF3 Ser379 and Ser396 as well as total IRF3 in RAW264.7-hSTING cells following 4 h treatment with 1% DMSO, 75 μM M04, 160 HAU/mL SeV, or transfection of cGAMP as indicated; (E) qPCR examining transcription of IFIT1 or Viperin following overnight treatment of parental RAW264.7 and RAW264.7-hSTING cells with 75 μM M04 ( n = 3). Data presented are mean fold changes ± SD of mRNA relative to cells treated with 1% DMSO.

    Article Snippet: Sources and concentrations of antibodies used against the following antigens are indicated in parentheses: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SC-51906; Santa Cruz) (1:10,000), IRF3 (4302; Cell Signaling), human phospho-IRF3 (76493; Abcam), mouse IRF3 (SC-9082; Santa Cruz), mouse phospho-S379 IRF3 (79945S; Cell Signaling), mouse phospho-S396 IRF3 (29047S; Cell Signaling), STING (13647S; Cell Signaling), phospho-S366 STING (19781S; Cell Signaling), TBK1 (3504S; Cell Signaling), phospho-TBK1 (5483S; Cell Signaling), NF-kB P65 (SC372; Santa Cruz), NF-kB P50 (3035; Cell Signaling), GM-130 (610823; BD Biosciences).

    Techniques: Expressing, Variant Assay, Reporter Assay, In Vivo, Injection, Transfection