anti irf3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti irf3
    TLR9 activation upregulated PD-L1 expression by promoting STAT3 Tyr705 phosphorylation in HCC cells. ( A ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 5, 10, or 20μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( B ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, or 10μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( C ) PD-L1 protein expression after treatment with ODN1585 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, and 10 μM; times: 0, 6, 12, 24, and 36 hours in 5μM) in Hepa1-6 cells. PD-L1 protein levels were analyzed by Western blotting. ( D ) PD-L1 + tumor cells were detected by flow cytometry after TLR9 agonist (Hep3B and Huh7 cells with ODN2216; Hepa1-6 cells with ODN1585) treatment with indicated concentration. (values are mean ± SD, *p < 0.05, **p < 0.01, ***p<0.001, NS indicates no significance). ( E ) PD-L1 expression after TLR9 overexpression in Huh7 cells. PD-L1 expression levels were analyzed by immunofluorescence. ( F ) PD-L1 protein expression after TLR9 overexpression in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( G ) PD-L1 protein expression in TLR9 knockdown or TLR9 rescue Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( H and I ) mRNA levels of PD-L1 in Hep3B ( H ) and Huh7 ( I ) cells measured by qRT-PCR after stimulation with different concentrations of ODN2216. (values are mean ± SD, *p < 0.05, **p < 0.01, NS indicates no significance) ( J ) TLR9 overexpression-induced PD-L1 expression after MYC, JUN, IRF1, <t>IRF3,</t> STAT1 or STAT3 silencing. PD-L1 mRNA expression in Huh7 cells was analyzed after TLR9 overexpression alone or in the presence of MYC-, JUN-, IRF1-, IRF3-, STAT1- or STAT3-specific siRNA or siRNA-NC. (values are mean ± SD, ***p<0.001). (K) p-STAT3 (Tyr705) levels in Hep3B cells after treatment with different concentrations of ODN2216 (a TLR9 agonist; ODN2216: 0, 2.5, 5, or 10μM) analyzed by Western blotting. ( L ) p-STAT3 (Tyr705) levels after TLR9 overexpression in Huh7 cells analyzed by Western blotting. ( M ) TLR9 overexpression-induced p-STAT3 (Tyr705) levels after TLR9 inhibition. p-STAT3 (Tyr705) levels were analyzed after TLR9 overexpression alone or in the presence of the TLR9 antagonist chloroquine diphosphate. ( N ) p-STAT3-induced PD-L1 levels after STAT3 inhibition. PD-L1 levels were analyzed after TLR9 overexpression alone or in the presence of the STAT3-specific small molecular inhibitor BP-1-102.
    Anti 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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    1) Product Images from "Hepatoma cell-intrinsic TLR9 activation induces immune escape through PD-L1 upregulation in hepatocellular carcinoma"

    Article Title: Hepatoma cell-intrinsic TLR9 activation induces immune escape through PD-L1 upregulation in hepatocellular carcinoma

    Journal: Theranostics

    doi: 10.7150/thno.44417

    TLR9 activation upregulated PD-L1 expression by promoting STAT3 Tyr705 phosphorylation in HCC cells. ( A ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 5, 10, or 20μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( B ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, or 10μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( C ) PD-L1 protein expression after treatment with ODN1585 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, and 10 μM; times: 0, 6, 12, 24, and 36 hours in 5μM) in Hepa1-6 cells. PD-L1 protein levels were analyzed by Western blotting. ( D ) PD-L1 + tumor cells were detected by flow cytometry after TLR9 agonist (Hep3B and Huh7 cells with ODN2216; Hepa1-6 cells with ODN1585) treatment with indicated concentration. (values are mean ± SD, *p < 0.05, **p < 0.01, ***p<0.001, NS indicates no significance). ( E ) PD-L1 expression after TLR9 overexpression in Huh7 cells. PD-L1 expression levels were analyzed by immunofluorescence. ( F ) PD-L1 protein expression after TLR9 overexpression in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( G ) PD-L1 protein expression in TLR9 knockdown or TLR9 rescue Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( H and I ) mRNA levels of PD-L1 in Hep3B ( H ) and Huh7 ( I ) cells measured by qRT-PCR after stimulation with different concentrations of ODN2216. (values are mean ± SD, *p < 0.05, **p < 0.01, NS indicates no significance) ( J ) TLR9 overexpression-induced PD-L1 expression after MYC, JUN, IRF1, IRF3, STAT1 or STAT3 silencing. PD-L1 mRNA expression in Huh7 cells was analyzed after TLR9 overexpression alone or in the presence of MYC-, JUN-, IRF1-, IRF3-, STAT1- or STAT3-specific siRNA or siRNA-NC. (values are mean ± SD, ***p<0.001). (K) p-STAT3 (Tyr705) levels in Hep3B cells after treatment with different concentrations of ODN2216 (a TLR9 agonist; ODN2216: 0, 2.5, 5, or 10μM) analyzed by Western blotting. ( L ) p-STAT3 (Tyr705) levels after TLR9 overexpression in Huh7 cells analyzed by Western blotting. ( M ) TLR9 overexpression-induced p-STAT3 (Tyr705) levels after TLR9 inhibition. p-STAT3 (Tyr705) levels were analyzed after TLR9 overexpression alone or in the presence of the TLR9 antagonist chloroquine diphosphate. ( N ) p-STAT3-induced PD-L1 levels after STAT3 inhibition. PD-L1 levels were analyzed after TLR9 overexpression alone or in the presence of the STAT3-specific small molecular inhibitor BP-1-102.
    Figure Legend Snippet: TLR9 activation upregulated PD-L1 expression by promoting STAT3 Tyr705 phosphorylation in HCC cells. ( A ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 5, 10, or 20μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( B ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, or 10μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( C ) PD-L1 protein expression after treatment with ODN1585 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, and 10 μM; times: 0, 6, 12, 24, and 36 hours in 5μM) in Hepa1-6 cells. PD-L1 protein levels were analyzed by Western blotting. ( D ) PD-L1 + tumor cells were detected by flow cytometry after TLR9 agonist (Hep3B and Huh7 cells with ODN2216; Hepa1-6 cells with ODN1585) treatment with indicated concentration. (values are mean ± SD, *p < 0.05, **p < 0.01, ***p<0.001, NS indicates no significance). ( E ) PD-L1 expression after TLR9 overexpression in Huh7 cells. PD-L1 expression levels were analyzed by immunofluorescence. ( F ) PD-L1 protein expression after TLR9 overexpression in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( G ) PD-L1 protein expression in TLR9 knockdown or TLR9 rescue Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( H and I ) mRNA levels of PD-L1 in Hep3B ( H ) and Huh7 ( I ) cells measured by qRT-PCR after stimulation with different concentrations of ODN2216. (values are mean ± SD, *p < 0.05, **p < 0.01, NS indicates no significance) ( J ) TLR9 overexpression-induced PD-L1 expression after MYC, JUN, IRF1, IRF3, STAT1 or STAT3 silencing. PD-L1 mRNA expression in Huh7 cells was analyzed after TLR9 overexpression alone or in the presence of MYC-, JUN-, IRF1-, IRF3-, STAT1- or STAT3-specific siRNA or siRNA-NC. (values are mean ± SD, ***p<0.001). (K) p-STAT3 (Tyr705) levels in Hep3B cells after treatment with different concentrations of ODN2216 (a TLR9 agonist; ODN2216: 0, 2.5, 5, or 10μM) analyzed by Western blotting. ( L ) p-STAT3 (Tyr705) levels after TLR9 overexpression in Huh7 cells analyzed by Western blotting. ( M ) TLR9 overexpression-induced p-STAT3 (Tyr705) levels after TLR9 inhibition. p-STAT3 (Tyr705) levels were analyzed after TLR9 overexpression alone or in the presence of the TLR9 antagonist chloroquine diphosphate. ( N ) p-STAT3-induced PD-L1 levels after STAT3 inhibition. PD-L1 levels were analyzed after TLR9 overexpression alone or in the presence of the STAT3-specific small molecular inhibitor BP-1-102.

    Techniques Used: Activation Assay, Expressing, Western Blot, Flow Cytometry, Concentration Assay, Over Expression, Immunofluorescence, Quantitative RT-PCR, Inhibition

    anti irf3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti irf3
    TLR9 activation upregulated PD-L1 expression by promoting STAT3 Tyr705 phosphorylation in HCC cells. ( A ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 5, 10, or 20μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( B ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, or 10μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( C ) PD-L1 protein expression after treatment with ODN1585 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, and 10 μM; times: 0, 6, 12, 24, and 36 hours in 5μM) in Hepa1-6 cells. PD-L1 protein levels were analyzed by Western blotting. ( D ) PD-L1 + tumor cells were detected by flow cytometry after TLR9 agonist (Hep3B and Huh7 cells with ODN2216; Hepa1-6 cells with ODN1585) treatment with indicated concentration. (values are mean ± SD, *p < 0.05, **p < 0.01, ***p<0.001, NS indicates no significance). ( E ) PD-L1 expression after TLR9 overexpression in Huh7 cells. PD-L1 expression levels were analyzed by immunofluorescence. ( F ) PD-L1 protein expression after TLR9 overexpression in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( G ) PD-L1 protein expression in TLR9 knockdown or TLR9 rescue Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( H and I ) mRNA levels of PD-L1 in Hep3B ( H ) and Huh7 ( I ) cells measured by qRT-PCR after stimulation with different concentrations of ODN2216. (values are mean ± SD, *p < 0.05, **p < 0.01, NS indicates no significance) ( J ) TLR9 overexpression-induced PD-L1 expression after MYC, JUN, IRF1, <t>IRF3,</t> STAT1 or STAT3 silencing. PD-L1 mRNA expression in Huh7 cells was analyzed after TLR9 overexpression alone or in the presence of MYC-, JUN-, IRF1-, IRF3-, STAT1- or STAT3-specific siRNA or siRNA-NC. (values are mean ± SD, ***p<0.001). (K) p-STAT3 (Tyr705) levels in Hep3B cells after treatment with different concentrations of ODN2216 (a TLR9 agonist; ODN2216: 0, 2.5, 5, or 10μM) analyzed by Western blotting. ( L ) p-STAT3 (Tyr705) levels after TLR9 overexpression in Huh7 cells analyzed by Western blotting. ( M ) TLR9 overexpression-induced p-STAT3 (Tyr705) levels after TLR9 inhibition. p-STAT3 (Tyr705) levels were analyzed after TLR9 overexpression alone or in the presence of the TLR9 antagonist chloroquine diphosphate. ( N ) p-STAT3-induced PD-L1 levels after STAT3 inhibition. PD-L1 levels were analyzed after TLR9 overexpression alone or in the presence of the STAT3-specific small molecular inhibitor BP-1-102.
    Anti 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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    1) Product Images from "Hepatoma cell-intrinsic TLR9 activation induces immune escape through PD-L1 upregulation in hepatocellular carcinoma"

    Article Title: Hepatoma cell-intrinsic TLR9 activation induces immune escape through PD-L1 upregulation in hepatocellular carcinoma

    Journal: Theranostics

    doi: 10.7150/thno.44417

    TLR9 activation upregulated PD-L1 expression by promoting STAT3 Tyr705 phosphorylation in HCC cells. ( A ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 5, 10, or 20μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( B ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, or 10μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( C ) PD-L1 protein expression after treatment with ODN1585 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, and 10 μM; times: 0, 6, 12, 24, and 36 hours in 5μM) in Hepa1-6 cells. PD-L1 protein levels were analyzed by Western blotting. ( D ) PD-L1 + tumor cells were detected by flow cytometry after TLR9 agonist (Hep3B and Huh7 cells with ODN2216; Hepa1-6 cells with ODN1585) treatment with indicated concentration. (values are mean ± SD, *p < 0.05, **p < 0.01, ***p<0.001, NS indicates no significance). ( E ) PD-L1 expression after TLR9 overexpression in Huh7 cells. PD-L1 expression levels were analyzed by immunofluorescence. ( F ) PD-L1 protein expression after TLR9 overexpression in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( G ) PD-L1 protein expression in TLR9 knockdown or TLR9 rescue Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( H and I ) mRNA levels of PD-L1 in Hep3B ( H ) and Huh7 ( I ) cells measured by qRT-PCR after stimulation with different concentrations of ODN2216. (values are mean ± SD, *p < 0.05, **p < 0.01, NS indicates no significance) ( J ) TLR9 overexpression-induced PD-L1 expression after MYC, JUN, IRF1, IRF3, STAT1 or STAT3 silencing. PD-L1 mRNA expression in Huh7 cells was analyzed after TLR9 overexpression alone or in the presence of MYC-, JUN-, IRF1-, IRF3-, STAT1- or STAT3-specific siRNA or siRNA-NC. (values are mean ± SD, ***p<0.001). (K) p-STAT3 (Tyr705) levels in Hep3B cells after treatment with different concentrations of ODN2216 (a TLR9 agonist; ODN2216: 0, 2.5, 5, or 10μM) analyzed by Western blotting. ( L ) p-STAT3 (Tyr705) levels after TLR9 overexpression in Huh7 cells analyzed by Western blotting. ( M ) TLR9 overexpression-induced p-STAT3 (Tyr705) levels after TLR9 inhibition. p-STAT3 (Tyr705) levels were analyzed after TLR9 overexpression alone or in the presence of the TLR9 antagonist chloroquine diphosphate. ( N ) p-STAT3-induced PD-L1 levels after STAT3 inhibition. PD-L1 levels were analyzed after TLR9 overexpression alone or in the presence of the STAT3-specific small molecular inhibitor BP-1-102.
    Figure Legend Snippet: TLR9 activation upregulated PD-L1 expression by promoting STAT3 Tyr705 phosphorylation in HCC cells. ( A ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 5, 10, or 20μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( B ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, or 10μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( C ) PD-L1 protein expression after treatment with ODN1585 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, and 10 μM; times: 0, 6, 12, 24, and 36 hours in 5μM) in Hepa1-6 cells. PD-L1 protein levels were analyzed by Western blotting. ( D ) PD-L1 + tumor cells were detected by flow cytometry after TLR9 agonist (Hep3B and Huh7 cells with ODN2216; Hepa1-6 cells with ODN1585) treatment with indicated concentration. (values are mean ± SD, *p < 0.05, **p < 0.01, ***p<0.001, NS indicates no significance). ( E ) PD-L1 expression after TLR9 overexpression in Huh7 cells. PD-L1 expression levels were analyzed by immunofluorescence. ( F ) PD-L1 protein expression after TLR9 overexpression in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( G ) PD-L1 protein expression in TLR9 knockdown or TLR9 rescue Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( H and I ) mRNA levels of PD-L1 in Hep3B ( H ) and Huh7 ( I ) cells measured by qRT-PCR after stimulation with different concentrations of ODN2216. (values are mean ± SD, *p < 0.05, **p < 0.01, NS indicates no significance) ( J ) TLR9 overexpression-induced PD-L1 expression after MYC, JUN, IRF1, IRF3, STAT1 or STAT3 silencing. PD-L1 mRNA expression in Huh7 cells was analyzed after TLR9 overexpression alone or in the presence of MYC-, JUN-, IRF1-, IRF3-, STAT1- or STAT3-specific siRNA or siRNA-NC. (values are mean ± SD, ***p<0.001). (K) p-STAT3 (Tyr705) levels in Hep3B cells after treatment with different concentrations of ODN2216 (a TLR9 agonist; ODN2216: 0, 2.5, 5, or 10μM) analyzed by Western blotting. ( L ) p-STAT3 (Tyr705) levels after TLR9 overexpression in Huh7 cells analyzed by Western blotting. ( M ) TLR9 overexpression-induced p-STAT3 (Tyr705) levels after TLR9 inhibition. p-STAT3 (Tyr705) levels were analyzed after TLR9 overexpression alone or in the presence of the TLR9 antagonist chloroquine diphosphate. ( N ) p-STAT3-induced PD-L1 levels after STAT3 inhibition. PD-L1 levels were analyzed after TLR9 overexpression alone or in the presence of the STAT3-specific small molecular inhibitor BP-1-102.

    Techniques Used: Activation Assay, Expressing, Western Blot, Flow Cytometry, Concentration Assay, Over Expression, Immunofluorescence, Quantitative RT-PCR, Inhibition

    irf3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc irf3
    Identification of RIG-I as the key sensor in type I IFN response in RPE. (a, b) RNA but not DNA could be sensed by RPE to induce type I IFN response, suggesting the lack of DNA sensors in unstimulated RPE. THP-1 and THP-1 cGAS KO cells were used as controls (a). Cells were treated with indicated inducers at 0.25 μ g/mL, with or without the Lipofectamine transfection reagent, and intracellular ISG15 was measured by ELISA 24 h after stimulation. Each bar represents biological replicates ( n = 3) and is indicated as mean ± SD. Note that ARPE-19 without transfection did not respond to most tested inducers except for poly(I:C). ∗ p < 0.05 compared with corresponded vehicle-treated groups. # p < 0.05 compared with same induces in parental cells. (c) Type I IFN response was induced via the <t>RIG-I–MAVS–IRF3</t> axis in ARPE-19 cells. ARPE-19 cells were cultured 10 d after reaching confluence for screening purposes. Different key nodes for the activation of the IFN pathway were knocked down using siRNAs (at least 3 different siRNAs for each gene). Cells were then treated with indicated nucleic acids, and the release of secreted IFN- β was measured by ELISA 24 h after stimulation. Similar results were observed in 3 independent experiments with different inducers (indicated in different lines) or different siRNAs (shown as different column); a single representative experiment is shown as an example. A heatmap was used to better visualize the percentage (%) change in the results. The efficiency of the mRNA knockdown was validated by qPCR and shown at the top in the form of heatmap. About ~70% inhibition was observed in most tested siRNAs.
    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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    1) Product Images from "Mechanism of Nucleic Acid Sensing in Retinal Pigment Epithelium (RPE): RIG-I Mediates Type I Interferon Response in Human RPE"

    Article Title: Mechanism of Nucleic Acid Sensing in Retinal Pigment Epithelium (RPE): RIG-I Mediates Type I Interferon Response in Human RPE

    Journal: Journal of Immunology Research

    doi: 10.1155/2021/9975628

    Identification of RIG-I as the key sensor in type I IFN response in RPE. (a, b) RNA but not DNA could be sensed by RPE to induce type I IFN response, suggesting the lack of DNA sensors in unstimulated RPE. THP-1 and THP-1 cGAS KO cells were used as controls (a). Cells were treated with indicated inducers at 0.25 μ g/mL, with or without the Lipofectamine transfection reagent, and intracellular ISG15 was measured by ELISA 24 h after stimulation. Each bar represents biological replicates ( n = 3) and is indicated as mean ± SD. Note that ARPE-19 without transfection did not respond to most tested inducers except for poly(I:C). ∗ p < 0.05 compared with corresponded vehicle-treated groups. # p < 0.05 compared with same induces in parental cells. (c) Type I IFN response was induced via the RIG-I–MAVS–IRF3 axis in ARPE-19 cells. ARPE-19 cells were cultured 10 d after reaching confluence for screening purposes. Different key nodes for the activation of the IFN pathway were knocked down using siRNAs (at least 3 different siRNAs for each gene). Cells were then treated with indicated nucleic acids, and the release of secreted IFN- β was measured by ELISA 24 h after stimulation. Similar results were observed in 3 independent experiments with different inducers (indicated in different lines) or different siRNAs (shown as different column); a single representative experiment is shown as an example. A heatmap was used to better visualize the percentage (%) change in the results. The efficiency of the mRNA knockdown was validated by qPCR and shown at the top in the form of heatmap. About ~70% inhibition was observed in most tested siRNAs.
    Figure Legend Snippet: Identification of RIG-I as the key sensor in type I IFN response in RPE. (a, b) RNA but not DNA could be sensed by RPE to induce type I IFN response, suggesting the lack of DNA sensors in unstimulated RPE. THP-1 and THP-1 cGAS KO cells were used as controls (a). Cells were treated with indicated inducers at 0.25 μ g/mL, with or without the Lipofectamine transfection reagent, and intracellular ISG15 was measured by ELISA 24 h after stimulation. Each bar represents biological replicates ( n = 3) and is indicated as mean ± SD. Note that ARPE-19 without transfection did not respond to most tested inducers except for poly(I:C). ∗ p < 0.05 compared with corresponded vehicle-treated groups. # p < 0.05 compared with same induces in parental cells. (c) Type I IFN response was induced via the RIG-I–MAVS–IRF3 axis in ARPE-19 cells. ARPE-19 cells were cultured 10 d after reaching confluence for screening purposes. Different key nodes for the activation of the IFN pathway were knocked down using siRNAs (at least 3 different siRNAs for each gene). Cells were then treated with indicated nucleic acids, and the release of secreted IFN- β was measured by ELISA 24 h after stimulation. Similar results were observed in 3 independent experiments with different inducers (indicated in different lines) or different siRNAs (shown as different column); a single representative experiment is shown as an example. A heatmap was used to better visualize the percentage (%) change in the results. The efficiency of the mRNA knockdown was validated by qPCR and shown at the top in the form of heatmap. About ~70% inhibition was observed in most tested siRNAs.

    Techniques Used: Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, Inhibition

    irf3 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc irf3 antibody
    cGAS-STING pathway was activated by arginine starvation. (A) Cells overexpressing cGAS-V5 were deprived for 72 h and stained with anti-V5 antibodies. Scale bars, 10 μm. (B) CWR22Rv1 cells were deprived for indicate timepoints, and endogenous <t>IRF3</t> was immunoprecipitated. IRF3 phosphorylation and TBK1 association were determined by immunoblots. Fold changes are listed below each blot. (C) IRF3 phosphorylation level was quantified and normalized with the IRF3. (n = 3, **p < 0.01). (D) RT-qPCR analysis of type I interferon expression in CWR22Rv1 cells starved for 72 h. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (E) IFNβ secretion by CWR22Rv1 cells after arginine starvation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (F) IFNβ promoter activity in CWR22Rv1 cells deprived for 72 h. (n = 3, *p < 0.05)
    Irf3 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
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    1) Product Images from "Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes"

    Article Title: Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes

    Journal: Theranostics

    doi: 10.7150/thno.54695

    cGAS-STING pathway was activated by arginine starvation. (A) Cells overexpressing cGAS-V5 were deprived for 72 h and stained with anti-V5 antibodies. Scale bars, 10 μm. (B) CWR22Rv1 cells were deprived for indicate timepoints, and endogenous IRF3 was immunoprecipitated. IRF3 phosphorylation and TBK1 association were determined by immunoblots. Fold changes are listed below each blot. (C) IRF3 phosphorylation level was quantified and normalized with the IRF3. (n = 3, **p < 0.01). (D) RT-qPCR analysis of type I interferon expression in CWR22Rv1 cells starved for 72 h. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (E) IFNβ secretion by CWR22Rv1 cells after arginine starvation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (F) IFNβ promoter activity in CWR22Rv1 cells deprived for 72 h. (n = 3, *p < 0.05)
    Figure Legend Snippet: cGAS-STING pathway was activated by arginine starvation. (A) Cells overexpressing cGAS-V5 were deprived for 72 h and stained with anti-V5 antibodies. Scale bars, 10 μm. (B) CWR22Rv1 cells were deprived for indicate timepoints, and endogenous IRF3 was immunoprecipitated. IRF3 phosphorylation and TBK1 association were determined by immunoblots. Fold changes are listed below each blot. (C) IRF3 phosphorylation level was quantified and normalized with the IRF3. (n = 3, **p < 0.01). (D) RT-qPCR analysis of type I interferon expression in CWR22Rv1 cells starved for 72 h. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (E) IFNβ secretion by CWR22Rv1 cells after arginine starvation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (F) IFNβ promoter activity in CWR22Rv1 cells deprived for 72 h. (n = 3, *p < 0.05)

    Techniques Used: Staining, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Expressing, Activity Assay

    Translocation of the cGAS downstream effectors. (A) Nuclear translocation of p65 was determined by subcellular fractionation in arginine-deprived CWR22Rv1 cells. (B) Nuclear p65 was quantified and normalized to lamin A/C. (n = 3, ***p < 0.001) (C) Immunostaining of p65 in CWR22Rv1 cells. Scale bars, 10 μm. (D) ChIP-qPCR analysis of IRF3 binding on the promoter regions of IFNA14 and IFNB1 in CWR22Rv1. (n = 3, *p < 0.05) (E) CWR22Rv1 cells overexpressing STING-V5 were subjected to arginine deprivation and stained with V5, calnexin and RCAS1 antibodies. Scale bars, 10 μm.
    Figure Legend Snippet: Translocation of the cGAS downstream effectors. (A) Nuclear translocation of p65 was determined by subcellular fractionation in arginine-deprived CWR22Rv1 cells. (B) Nuclear p65 was quantified and normalized to lamin A/C. (n = 3, ***p < 0.001) (C) Immunostaining of p65 in CWR22Rv1 cells. Scale bars, 10 μm. (D) ChIP-qPCR analysis of IRF3 binding on the promoter regions of IFNA14 and IFNB1 in CWR22Rv1. (n = 3, *p < 0.05) (E) CWR22Rv1 cells overexpressing STING-V5 were subjected to arginine deprivation and stained with V5, calnexin and RCAS1 antibodies. Scale bars, 10 μm.

    Techniques Used: Translocation Assay, Fractionation, Immunostaining, Binding Assay, Staining

    irf3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc irf3
    cGAS-STING pathway was activated by arginine starvation. (A) Cells overexpressing cGAS-V5 were deprived for 72 h and stained with anti-V5 antibodies. Scale bars, 10 μm. (B) CWR22Rv1 cells were deprived for indicate timepoints, and endogenous <t>IRF3</t> was immunoprecipitated. IRF3 phosphorylation and TBK1 association were determined by immunoblots. Fold changes are listed below each blot. (C) IRF3 phosphorylation level was quantified and normalized with the IRF3. (n = 3, **p < 0.01). (D) RT-qPCR analysis of type I interferon expression in CWR22Rv1 cells starved for 72 h. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (E) IFNβ secretion by CWR22Rv1 cells after arginine starvation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (F) IFNβ promoter activity in CWR22Rv1 cells deprived for 72 h. (n = 3, *p < 0.05)
    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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    1) Product Images from "Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes"

    Article Title: Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes

    Journal: Theranostics

    doi: 10.7150/thno.54695

    cGAS-STING pathway was activated by arginine starvation. (A) Cells overexpressing cGAS-V5 were deprived for 72 h and stained with anti-V5 antibodies. Scale bars, 10 μm. (B) CWR22Rv1 cells were deprived for indicate timepoints, and endogenous IRF3 was immunoprecipitated. IRF3 phosphorylation and TBK1 association were determined by immunoblots. Fold changes are listed below each blot. (C) IRF3 phosphorylation level was quantified and normalized with the IRF3. (n = 3, **p < 0.01). (D) RT-qPCR analysis of type I interferon expression in CWR22Rv1 cells starved for 72 h. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (E) IFNβ secretion by CWR22Rv1 cells after arginine starvation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (F) IFNβ promoter activity in CWR22Rv1 cells deprived for 72 h. (n = 3, *p < 0.05)
    Figure Legend Snippet: cGAS-STING pathway was activated by arginine starvation. (A) Cells overexpressing cGAS-V5 were deprived for 72 h and stained with anti-V5 antibodies. Scale bars, 10 μm. (B) CWR22Rv1 cells were deprived for indicate timepoints, and endogenous IRF3 was immunoprecipitated. IRF3 phosphorylation and TBK1 association were determined by immunoblots. Fold changes are listed below each blot. (C) IRF3 phosphorylation level was quantified and normalized with the IRF3. (n = 3, **p < 0.01). (D) RT-qPCR analysis of type I interferon expression in CWR22Rv1 cells starved for 72 h. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (E) IFNβ secretion by CWR22Rv1 cells after arginine starvation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (F) IFNβ promoter activity in CWR22Rv1 cells deprived for 72 h. (n = 3, *p < 0.05)

    Techniques Used: Staining, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Expressing, Activity Assay

    Translocation of the cGAS downstream effectors. (A) Nuclear translocation of p65 was determined by subcellular fractionation in arginine-deprived CWR22Rv1 cells. (B) Nuclear p65 was quantified and normalized to lamin A/C. (n = 3, ***p < 0.001) (C) Immunostaining of p65 in CWR22Rv1 cells. Scale bars, 10 μm. (D) ChIP-qPCR analysis of IRF3 binding on the promoter regions of IFNA14 and IFNB1 in CWR22Rv1. (n = 3, *p < 0.05) (E) CWR22Rv1 cells overexpressing STING-V5 were subjected to arginine deprivation and stained with V5, calnexin and RCAS1 antibodies. Scale bars, 10 μm.
    Figure Legend Snippet: Translocation of the cGAS downstream effectors. (A) Nuclear translocation of p65 was determined by subcellular fractionation in arginine-deprived CWR22Rv1 cells. (B) Nuclear p65 was quantified and normalized to lamin A/C. (n = 3, ***p < 0.001) (C) Immunostaining of p65 in CWR22Rv1 cells. Scale bars, 10 μm. (D) ChIP-qPCR analysis of IRF3 binding on the promoter regions of IFNA14 and IFNB1 in CWR22Rv1. (n = 3, *p < 0.05) (E) CWR22Rv1 cells overexpressing STING-V5 were subjected to arginine deprivation and stained with V5, calnexin and RCAS1 antibodies. Scale bars, 10 μm.

    Techniques Used: Translocation Assay, Fractionation, Immunostaining, Binding Assay, Staining

    rabbit irf3 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit irf3 antibody
    Rabbit Irf3 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
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    ifr3 11904s  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ifr3 11904s
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    anti irf3 11904 antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti irf3 11904 antibodies
    (A) Human cGAS mutants used for functional analyses. Human cGAS is shown as a ribbon model, with the same coloring as in . Mutated residues are shown as red ball-and-stick models. (B) cGAS-induced phosphorylation of TBK1, <t>IRF3,</t> and STING. cGAS WT or mutants were expressed in HEK293T cells stably expressing human STING. Cell lysates were analyzed by western blotting, using the indicated antibodies. The asterisk indicates phosphorylated STING. (C) Reporter assays for IFN-β (left panel) and NF-κB (right panel). The cell lysates are the same as in (B), except for the co-expression with reporter plasmids, and were measured for luciferase activities. Luciferase activities are shown as mean ± s.d. (n = 3). (D) Induction of IFN-β and A20 by human cGAS and its mutants. The relative mRNA expression levels of IFN-β (left) and A20 (right) were analyzed by Real-Time PCR using total RNAs isolated from the cells shown in (B). Relative expression levels are shown as mean ± s.d. (n = 3). (E) Immunoblotting for cGAS-induced phosphorylation (left panel), reporter assays for IFN-β (upper panel) and NF-κB (lower panel), the same as (B) and (C), respectively. (F) Pull-down experiment between biotinylated-ISD and human cGAS mutants. Human cGAS mutants were expressed and purified from E. coli , and mixed with Streptavidin beads in the presence or absence of biotinylated-ISD. Bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE.
    Anti Irf3 11904 Antibodies, 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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    1) Product Images from "Structural and Functional Analyses of DNA-Sensing and Immune Activation by Human cGAS"

    Article Title: Structural and Functional Analyses of DNA-Sensing and Immune Activation by Human cGAS

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076983

    (A) Human cGAS mutants used for functional analyses. Human cGAS is shown as a ribbon model, with the same coloring as in . Mutated residues are shown as red ball-and-stick models. (B) cGAS-induced phosphorylation of TBK1, IRF3, and STING. cGAS WT or mutants were expressed in HEK293T cells stably expressing human STING. Cell lysates were analyzed by western blotting, using the indicated antibodies. The asterisk indicates phosphorylated STING. (C) Reporter assays for IFN-β (left panel) and NF-κB (right panel). The cell lysates are the same as in (B), except for the co-expression with reporter plasmids, and were measured for luciferase activities. Luciferase activities are shown as mean ± s.d. (n = 3). (D) Induction of IFN-β and A20 by human cGAS and its mutants. The relative mRNA expression levels of IFN-β (left) and A20 (right) were analyzed by Real-Time PCR using total RNAs isolated from the cells shown in (B). Relative expression levels are shown as mean ± s.d. (n = 3). (E) Immunoblotting for cGAS-induced phosphorylation (left panel), reporter assays for IFN-β (upper panel) and NF-κB (lower panel), the same as (B) and (C), respectively. (F) Pull-down experiment between biotinylated-ISD and human cGAS mutants. Human cGAS mutants were expressed and purified from E. coli , and mixed with Streptavidin beads in the presence or absence of biotinylated-ISD. Bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE.
    Figure Legend Snippet: (A) Human cGAS mutants used for functional analyses. Human cGAS is shown as a ribbon model, with the same coloring as in . Mutated residues are shown as red ball-and-stick models. (B) cGAS-induced phosphorylation of TBK1, IRF3, and STING. cGAS WT or mutants were expressed in HEK293T cells stably expressing human STING. Cell lysates were analyzed by western blotting, using the indicated antibodies. The asterisk indicates phosphorylated STING. (C) Reporter assays for IFN-β (left panel) and NF-κB (right panel). The cell lysates are the same as in (B), except for the co-expression with reporter plasmids, and were measured for luciferase activities. Luciferase activities are shown as mean ± s.d. (n = 3). (D) Induction of IFN-β and A20 by human cGAS and its mutants. The relative mRNA expression levels of IFN-β (left) and A20 (right) were analyzed by Real-Time PCR using total RNAs isolated from the cells shown in (B). Relative expression levels are shown as mean ± s.d. (n = 3). (E) Immunoblotting for cGAS-induced phosphorylation (left panel), reporter assays for IFN-β (upper panel) and NF-κB (lower panel), the same as (B) and (C), respectively. (F) Pull-down experiment between biotinylated-ISD and human cGAS mutants. Human cGAS mutants were expressed and purified from E. coli , and mixed with Streptavidin beads in the presence or absence of biotinylated-ISD. Bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE.

    Techniques Used: Functional Assay, Stable Transfection, Expressing, Western Blot, Luciferase, Real-time Polymerase Chain Reaction, Isolation, Purification, SDS Page

    anti irf3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti irf3
    A, B Overlays of images of A549 cells at three time points post infection (p.i.) with RSV at an MOI of 0.01, immunostained for: RSV proteins (polyclonal antibody) – magenta, <t>IRF3</t> – red, (intracellular) IFNβ – yellow (only in panel A), phospho-Tyr701 STAT1 (p-STAT1) – green (only in panel B). The 2 h-long treatment with brefeldin A (BFA, in panel A) prevented secretion of IFNβ produced during the treatment. When phosphorylated, IRF3 translocates to the nucleus giving a discernible signal. White dotted lines are nuclear outlines determined based on DAPI counterstaining (channel not shown). Scale bars, 50 µm. C Snapshots from a simulation of infection at an MOI of 0.01 in a compact monolayer of cells (subpanels show fragments of a simulated 100×100 lattice). The color key corresponds to pseudocolors of immunostained proteins used in panels A, B (concentration of IFNβ in the lower subcompartment is indicated at hexagon borders; yellow color in nuclei results from mixing of red and green).
    Anti 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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    1) Product Images from "Antagonism between viral infection and innate immunity at the single-cell level"

    Article Title: Antagonism between viral infection and innate immunity at the single-cell level

    Journal: bioRxiv

    doi: 10.1101/2022.11.18.517110

    A, B Overlays of images of A549 cells at three time points post infection (p.i.) with RSV at an MOI of 0.01, immunostained for: RSV proteins (polyclonal antibody) – magenta, IRF3 – red, (intracellular) IFNβ – yellow (only in panel A), phospho-Tyr701 STAT1 (p-STAT1) – green (only in panel B). The 2 h-long treatment with brefeldin A (BFA, in panel A) prevented secretion of IFNβ produced during the treatment. When phosphorylated, IRF3 translocates to the nucleus giving a discernible signal. White dotted lines are nuclear outlines determined based on DAPI counterstaining (channel not shown). Scale bars, 50 µm. C Snapshots from a simulation of infection at an MOI of 0.01 in a compact monolayer of cells (subpanels show fragments of a simulated 100×100 lattice). The color key corresponds to pseudocolors of immunostained proteins used in panels A, B (concentration of IFNβ in the lower subcompartment is indicated at hexagon borders; yellow color in nuclei results from mixing of red and green).
    Figure Legend Snippet: A, B Overlays of images of A549 cells at three time points post infection (p.i.) with RSV at an MOI of 0.01, immunostained for: RSV proteins (polyclonal antibody) – magenta, IRF3 – red, (intracellular) IFNβ – yellow (only in panel A), phospho-Tyr701 STAT1 (p-STAT1) – green (only in panel B). The 2 h-long treatment with brefeldin A (BFA, in panel A) prevented secretion of IFNβ produced during the treatment. When phosphorylated, IRF3 translocates to the nucleus giving a discernible signal. White dotted lines are nuclear outlines determined based on DAPI counterstaining (channel not shown). Scale bars, 50 µm. C Snapshots from a simulation of infection at an MOI of 0.01 in a compact monolayer of cells (subpanels show fragments of a simulated 100×100 lattice). The color key corresponds to pseudocolors of immunostained proteins used in panels A, B (concentration of IFNβ in the lower subcompartment is indicated at hexagon borders; yellow color in nuclei results from mixing of red and green).

    Techniques Used: Infection, Produced, Concentration Assay

    A, B A549 cells 10, 16, and 24 hours post infection (p.i.) with RSV at an MOI of 1. IRF3 – red (intracellular, only in panel A), RSV – magenta (only in panel B), p-STAT1 – green. White dotted lines are nuclear outlines determined based on DAPI counterstaining (channel not shown). Scale bars, 50 µm. C Snapshots from a simulation of infection at an MOI of 1 in a compact monolayer of cells (subpanels show small fragments of a simulated 100×100 lattice).
    Figure Legend Snippet: A, B A549 cells 10, 16, and 24 hours post infection (p.i.) with RSV at an MOI of 1. IRF3 – red (intracellular, only in panel A), RSV – magenta (only in panel B), p-STAT1 – green. White dotted lines are nuclear outlines determined based on DAPI counterstaining (channel not shown). Scale bars, 50 µm. C Snapshots from a simulation of infection at an MOI of 1 in a compact monolayer of cells (subpanels show small fragments of a simulated 100×100 lattice).

    Techniques Used: Infection

    A A549 cells 24 h post infection (p.i.) with RSV at an MOI of 0.1, immunostained for RSV proteins – magenta, p-STAT1 – green, and IRF3 – red; left to right, incremental overlays. White dotted lines are nuclear outlines determined based on DAPI counterstaining (channel not shown). Scale bar, 50 µm. B Distributions of the mean nuclear p-STAT1 intensities in cells either expressing or not expressing RSV proteins (top subpanel, ‘same cells’) and in cells not expressing RSV proteins but either having or not a neighboring cell expressing RSV proteins (bottom subpanel, ‘neighboring cells’). Pairs of histograms, cell counts, and signed Kolmogorov–Smirnov (sKS) statistics are given for a representative experiment of an MOI of 0.1 (marked in panel C with an arrow). C Dependence of the ‘same cells’ sKS(pSTATs | RSV proteins) statistics and ‘neighboring cells’ sKS(pSTATs | RSV proteins) statistics on the fraction of RSV proteins-expressing cells at 24 h p.i. Marker sizes correspond to the number of cells quantified in each experiment. Experimental results are juxtaposed with model predictions (dotted lines enveloped within 95% CrI computed for n = 2500 cells). D ‘Same cells’ sKS(pSTATs | RSV proteins) over the period of two days post infection at an MOI of 0.1, according to the model. The dashed lines were obtained at different strengths of the vProteins ⊣ pSTATs inhibition. E Model-based analysis of the influence of the vProteins ⊣ pSTATs inhibition: ‘Same cells’ sKS(pSTATs | RSV proteins) (upper subpanel, at 24 h p.i.), average ISGs and vProteins states (middle subpanel, at 48 h p.i.), average level of extracellular interferon IFNe (lower subpanel, at 48 h p.i.).
    Figure Legend Snippet: A A549 cells 24 h post infection (p.i.) with RSV at an MOI of 0.1, immunostained for RSV proteins – magenta, p-STAT1 – green, and IRF3 – red; left to right, incremental overlays. White dotted lines are nuclear outlines determined based on DAPI counterstaining (channel not shown). Scale bar, 50 µm. B Distributions of the mean nuclear p-STAT1 intensities in cells either expressing or not expressing RSV proteins (top subpanel, ‘same cells’) and in cells not expressing RSV proteins but either having or not a neighboring cell expressing RSV proteins (bottom subpanel, ‘neighboring cells’). Pairs of histograms, cell counts, and signed Kolmogorov–Smirnov (sKS) statistics are given for a representative experiment of an MOI of 0.1 (marked in panel C with an arrow). C Dependence of the ‘same cells’ sKS(pSTATs | RSV proteins) statistics and ‘neighboring cells’ sKS(pSTATs | RSV proteins) statistics on the fraction of RSV proteins-expressing cells at 24 h p.i. Marker sizes correspond to the number of cells quantified in each experiment. Experimental results are juxtaposed with model predictions (dotted lines enveloped within 95% CrI computed for n = 2500 cells). D ‘Same cells’ sKS(pSTATs | RSV proteins) over the period of two days post infection at an MOI of 0.1, according to the model. The dashed lines were obtained at different strengths of the vProteins ⊣ pSTATs inhibition. E Model-based analysis of the influence of the vProteins ⊣ pSTATs inhibition: ‘Same cells’ sKS(pSTATs | RSV proteins) (upper subpanel, at 24 h p.i.), average ISGs and vProteins states (middle subpanel, at 48 h p.i.), average level of extracellular interferon IFNe (lower subpanel, at 48 h p.i.).

    Techniques Used: Infection, Expressing, Marker, Inhibition

    A Incremental overlays of images of A549 cells 24 h post infection (p.i.) with RSV at an MOI of 0.1, immunostained for IRF3 – red, RSV – magenta, and p-STAT1 – green. White dotted lines are nuclear outlines determined based on DAPI counterstaining (channel not shown). Scale bar, 50 µm. B Conditional probability P (pIRF3 | RSV proteins) as a function of the percentage of RSV proteins-expressing cells. Experiments (at 16, 20, 24, 36, 40, 48 h p.i.) – disks, model (at 16, 24, 48 h p.i.) – lines (line points were computed for a specific MOI, resulting in a given proportion of RSV-positive cells; results are averages over 1000 stochastic simulations on the 100×100 lattice). Dashed lines show model predictions (at 24 h p.i.) without all 5 inhibitions or with 10 times stronger inhibitions (as shown in the figure). Dotted lines show model predictions (at 24 h p.i.) without the vProteins ⊣ IRF3 inhibition or with 10 times stronger inhibition. C Conditional probability P (RSV proteins | pIRF3), notation as in panel B. D Proportion of the (IRF3 & vProtein)-positive cells (left) and IRF3-positive cells (right) as a function of time for a default strength of the vProteins ⊣ pIRF3 inhibition, no inhibition, and the inhibition 10 times stronger than default.
    Figure Legend Snippet: A Incremental overlays of images of A549 cells 24 h post infection (p.i.) with RSV at an MOI of 0.1, immunostained for IRF3 – red, RSV – magenta, and p-STAT1 – green. White dotted lines are nuclear outlines determined based on DAPI counterstaining (channel not shown). Scale bar, 50 µm. B Conditional probability P (pIRF3 | RSV proteins) as a function of the percentage of RSV proteins-expressing cells. Experiments (at 16, 20, 24, 36, 40, 48 h p.i.) – disks, model (at 16, 24, 48 h p.i.) – lines (line points were computed for a specific MOI, resulting in a given proportion of RSV-positive cells; results are averages over 1000 stochastic simulations on the 100×100 lattice). Dashed lines show model predictions (at 24 h p.i.) without all 5 inhibitions or with 10 times stronger inhibitions (as shown in the figure). Dotted lines show model predictions (at 24 h p.i.) without the vProteins ⊣ IRF3 inhibition or with 10 times stronger inhibition. C Conditional probability P (RSV proteins | pIRF3), notation as in panel B. D Proportion of the (IRF3 & vProtein)-positive cells (left) and IRF3-positive cells (right) as a function of time for a default strength of the vProteins ⊣ pIRF3 inhibition, no inhibition, and the inhibition 10 times stronger than default.

    Techniques Used: Infection, Expressing, Inhibition

    A Incremental overlays of images of A549 cells 26 hours post infection (h p.i.) with RSV at MOI 0.1, immunostained for IFNβ – yellow, RSV – magenta, and IRF3 – red. The 2 hour-long treatment with brefeldin A (BFA) prevents secretion of IFNβ produced by some cells during the treatment. White dotted lines are nuclear outlines determined based on DAPI counterstaining (channel not shown). Scale bar, 50 µm. B Conditional probability P (IFN | RSV proteins) as a function of the percentage of RSV proteins-expressing cells. Experiments (at 16, 20, 24, 36, 40, 48 h p.i.) – disks, model (at 16, 24, 48 h p.i.) – lines (line points were computed for a specific MOI, resulting in a given proportion of RSV-positive cells; results are averages over 1000 stochastic simulations on the 100×100 lattice). Dashed lines show model predictions (at 24 h p.i.) without all 5 inhibitions or with 10 times stronger inhibitions (as shown in the figure). Dotted lines show model predictions (at 24 h p.i.) without vProteins ⊣ pIRF3 and vProteins ⊣ IFNi inhibitions, or with these two inhibitions 10 times stronger. C Conditional probability P (RSV proteins | IFN), notation as in panel B.
    Figure Legend Snippet: A Incremental overlays of images of A549 cells 26 hours post infection (h p.i.) with RSV at MOI 0.1, immunostained for IFNβ – yellow, RSV – magenta, and IRF3 – red. The 2 hour-long treatment with brefeldin A (BFA) prevents secretion of IFNβ produced by some cells during the treatment. White dotted lines are nuclear outlines determined based on DAPI counterstaining (channel not shown). Scale bar, 50 µm. B Conditional probability P (IFN | RSV proteins) as a function of the percentage of RSV proteins-expressing cells. Experiments (at 16, 20, 24, 36, 40, 48 h p.i.) – disks, model (at 16, 24, 48 h p.i.) – lines (line points were computed for a specific MOI, resulting in a given proportion of RSV-positive cells; results are averages over 1000 stochastic simulations on the 100×100 lattice). Dashed lines show model predictions (at 24 h p.i.) without all 5 inhibitions or with 10 times stronger inhibitions (as shown in the figure). Dotted lines show model predictions (at 24 h p.i.) without vProteins ⊣ pIRF3 and vProteins ⊣ IFNi inhibitions, or with these two inhibitions 10 times stronger. C Conditional probability P (RSV proteins | IFN), notation as in panel B.

    Techniques Used: Infection, Produced, Expressing

    A Simplified scheme of antagonistic interaction present in the model. B–E ( B ) Fraction of cells producing IFNe ( IFNi = 3 ) and vProteins ( vProteins > 0 ) simultaneously, ( C ) fraction of cells producing IFNe , ( D ) average activation of pSTATs , ( E ) average activation of ISGs , ( F ) fraction of cells producing vProteins ; all as a function of time for an MOI of 0.01 for default inhibition strengths (black line), inhibitions vProteins ⊣ IRF3 and vProteins ⊣ IFNi 10 times stronger (thick line), and turned off (dashed, dotted and dash-dotted lines). G Change of the average level of vProteins and of the fraction of cells producing IFNe as a function of the ISGs ⊣ vProteins inhibition, at 48 h p.i. at an MOI of 0.01. H Change of the average level of vProteins and of the fraction of cells producing IFNe as a function of the ISGs ⊣ vRNA inhibition, at 48 h p.i. at an MOI of 0.01.
    Figure Legend Snippet: A Simplified scheme of antagonistic interaction present in the model. B–E ( B ) Fraction of cells producing IFNe ( IFNi = 3 ) and vProteins ( vProteins > 0 ) simultaneously, ( C ) fraction of cells producing IFNe , ( D ) average activation of pSTATs , ( E ) average activation of ISGs , ( F ) fraction of cells producing vProteins ; all as a function of time for an MOI of 0.01 for default inhibition strengths (black line), inhibitions vProteins ⊣ IRF3 and vProteins ⊣ IFNi 10 times stronger (thick line), and turned off (dashed, dotted and dash-dotted lines). G Change of the average level of vProteins and of the fraction of cells producing IFNe as a function of the ISGs ⊣ vProteins inhibition, at 48 h p.i. at an MOI of 0.01. H Change of the average level of vProteins and of the fraction of cells producing IFNe as a function of the ISGs ⊣ vRNA inhibition, at 48 h p.i. at an MOI of 0.01.

    Techniques Used: Activation Assay, Inhibition

    rabbit monoclonal anti irf3 d6i4c  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc rabbit monoclonal anti irf3 d6i4c
    a , Schematic diagram of the CTT of human STING (hSTING) and sequence logo of the CTT as indicated from 50 species. b , HeLa STING-knockout (KO) cells transfected with Flag-tagged STING ΔCΤΤ (Δ1–341), wild-type (WT) STING, STING E (E360A), STING LI (L364A/I365A) or STING ELI (E360A/L364A/I365A) were treated with diABZI for 2 h. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. c , WCL and extracted CCV fractions from HeLa STING KO cells reconstituted with Flag-tagged wild-type STING or STING LI (L364A/I365A) and treated or not with diABZI were analysed by western blot. CHC was used as a loading control. d , Glutathione sepharose pull-down assays of wild-type LBD-STING or LBD-STING ELI by glutathione S -transferase (GST)-tagged AP-1 core with or without ARF1. e , HeLa STING cells transfected with NC siRNA or siRNAs against TBK1 or <t>IRF3</t> were treated with or without diABZI. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. GAPDH was used as a loading control. f , HeLa wild-type cells, HeLa TBK1 KO cells and HeLa IRF3 KO cells stimulated with diABZI for 0, 1, 2 or 4 h were analysed by western blot. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition. Vinculin was used as a loading control. g , Bio-layer interferometry binding studies of LBD-STING (top) or TBK1-phosphorylated LBD-STING (pSTING) (bottom) with AP-1 ΔμCTD. The right graphs show the binding affinity of STING (top) and pSTING (bottom). One representative example of at least three ( b , d , f ) or two ( c , e , g ) independent experiments is shown.
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    Images

    1) Product Images from "Clathrin-associated AP-1 controls termination of STING signalling"

    Article Title: Clathrin-associated AP-1 controls termination of STING signalling

    Journal: Nature

    doi: 10.1038/s41586-022-05354-0

    a , Schematic diagram of the CTT of human STING (hSTING) and sequence logo of the CTT as indicated from 50 species. b , HeLa STING-knockout (KO) cells transfected with Flag-tagged STING ΔCΤΤ (Δ1–341), wild-type (WT) STING, STING E (E360A), STING LI (L364A/I365A) or STING ELI (E360A/L364A/I365A) were treated with diABZI for 2 h. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. c , WCL and extracted CCV fractions from HeLa STING KO cells reconstituted with Flag-tagged wild-type STING or STING LI (L364A/I365A) and treated or not with diABZI were analysed by western blot. CHC was used as a loading control. d , Glutathione sepharose pull-down assays of wild-type LBD-STING or LBD-STING ELI by glutathione S -transferase (GST)-tagged AP-1 core with or without ARF1. e , HeLa STING cells transfected with NC siRNA or siRNAs against TBK1 or IRF3 were treated with or without diABZI. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. GAPDH was used as a loading control. f , HeLa wild-type cells, HeLa TBK1 KO cells and HeLa IRF3 KO cells stimulated with diABZI for 0, 1, 2 or 4 h were analysed by western blot. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition. Vinculin was used as a loading control. g , Bio-layer interferometry binding studies of LBD-STING (top) or TBK1-phosphorylated LBD-STING (pSTING) (bottom) with AP-1 ΔμCTD. The right graphs show the binding affinity of STING (top) and pSTING (bottom). One representative example of at least three ( b , d , f ) or two ( c , e , g ) independent experiments is shown.
    Figure Legend Snippet: a , Schematic diagram of the CTT of human STING (hSTING) and sequence logo of the CTT as indicated from 50 species. b , HeLa STING-knockout (KO) cells transfected with Flag-tagged STING ΔCΤΤ (Δ1–341), wild-type (WT) STING, STING E (E360A), STING LI (L364A/I365A) or STING ELI (E360A/L364A/I365A) were treated with diABZI for 2 h. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. c , WCL and extracted CCV fractions from HeLa STING KO cells reconstituted with Flag-tagged wild-type STING or STING LI (L364A/I365A) and treated or not with diABZI were analysed by western blot. CHC was used as a loading control. d , Glutathione sepharose pull-down assays of wild-type LBD-STING or LBD-STING ELI by glutathione S -transferase (GST)-tagged AP-1 core with or without ARF1. e , HeLa STING cells transfected with NC siRNA or siRNAs against TBK1 or IRF3 were treated with or without diABZI. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. GAPDH was used as a loading control. f , HeLa wild-type cells, HeLa TBK1 KO cells and HeLa IRF3 KO cells stimulated with diABZI for 0, 1, 2 or 4 h were analysed by western blot. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition. Vinculin was used as a loading control. g , Bio-layer interferometry binding studies of LBD-STING (top) or TBK1-phosphorylated LBD-STING (pSTING) (bottom) with AP-1 ΔμCTD. The right graphs show the binding affinity of STING (top) and pSTING (bottom). One representative example of at least three ( b , d , f ) or two ( c , e , g ) independent experiments is shown.

    Techniques Used: Sequencing, Knock-Out, Transfection, Immunoprecipitation, Western Blot, Binding Assay

    anti irf3  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc anti irf3
    a, Nimbolide treatment induces PARP1 trapping. UWB1 cells were treated with or without nimbolide (1 μM for 48 hrs). The chromatin-bound fraction was isolated from these cells, and was subject to immunoblotting experiments using the indicated antibodies. b, Nimbolide treatment induces DNA damage response. HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The cell lysates were subject to immunoblotting experiments using the indicated antibodies. c, Nimbolide treatment induces the formation of cytosolic dsDNA and micronuclei. HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). DAPI (blue) was used to visualize the nuclei. Arrows indicate cytosolic dsDNA and micronuclei. Scale Bars, 10 μm. d, Nimbolide treatment induces the phosphorylation of TBK1. HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The level of pS172 TBK1 (p-TBK1, Green) was detected using the immunofluorescence assay. DAPI (blue) was used to visualize the nuclei. Scale Bars, 10 μm. e, Nimbolide treatment induces the phosphorylation of <t>IRF3.</t> HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The level of pS396 IRF3 (p-IRF3, Green) was detected using the immunofluorescence assay. DAPI (blue) was used to visualize the nuclei. Scale Bars, 10 μm. f, qRT-PCR analyses of IFN-β, CXCL10, or CCL5 in HeLa cells treated with or without nimbolide (1 μM for 48 hrs). g, Nimbolide treatment induces the expression of PD-L1. UWB1 cells were treated with or without nimbolide (1 μM for 48 hrs). PD-L1 expression was detected using the immunoblot assay. h, RNF114-KO abrogates the nimbolide-induced TBK1 phosphorylation. Control (RNF114-WT) and RNF114-KO HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The whole cell lysates were subject to immunoblot experiments using the indicated antibodies. i, PARP1-KO abrogates the nimbolide-induced TBK1 phosphorylation. Control (PARP1-WT) and PARP1-KO HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The whole cell lysates were subject to immunoblot experiments using the indicated antibodies.
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    Images

    1) Product Images from "Nimbolide Targets RNF114 to Induce the Trapping of PARP1 and Poly-ADP-Ribosylation-Dependent DNA Repair Factors"

    Article Title: Nimbolide Targets RNF114 to Induce the Trapping of PARP1 and Poly-ADP-Ribosylation-Dependent DNA Repair Factors

    Journal: bioRxiv

    doi: 10.1101/2022.10.04.510815

    a, Nimbolide treatment induces PARP1 trapping. UWB1 cells were treated with or without nimbolide (1 μM for 48 hrs). The chromatin-bound fraction was isolated from these cells, and was subject to immunoblotting experiments using the indicated antibodies. b, Nimbolide treatment induces DNA damage response. HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The cell lysates were subject to immunoblotting experiments using the indicated antibodies. c, Nimbolide treatment induces the formation of cytosolic dsDNA and micronuclei. HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). DAPI (blue) was used to visualize the nuclei. Arrows indicate cytosolic dsDNA and micronuclei. Scale Bars, 10 μm. d, Nimbolide treatment induces the phosphorylation of TBK1. HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The level of pS172 TBK1 (p-TBK1, Green) was detected using the immunofluorescence assay. DAPI (blue) was used to visualize the nuclei. Scale Bars, 10 μm. e, Nimbolide treatment induces the phosphorylation of IRF3. HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The level of pS396 IRF3 (p-IRF3, Green) was detected using the immunofluorescence assay. DAPI (blue) was used to visualize the nuclei. Scale Bars, 10 μm. f, qRT-PCR analyses of IFN-β, CXCL10, or CCL5 in HeLa cells treated with or without nimbolide (1 μM for 48 hrs). g, Nimbolide treatment induces the expression of PD-L1. UWB1 cells were treated with or without nimbolide (1 μM for 48 hrs). PD-L1 expression was detected using the immunoblot assay. h, RNF114-KO abrogates the nimbolide-induced TBK1 phosphorylation. Control (RNF114-WT) and RNF114-KO HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The whole cell lysates were subject to immunoblot experiments using the indicated antibodies. i, PARP1-KO abrogates the nimbolide-induced TBK1 phosphorylation. Control (PARP1-WT) and PARP1-KO HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The whole cell lysates were subject to immunoblot experiments using the indicated antibodies.
    Figure Legend Snippet: a, Nimbolide treatment induces PARP1 trapping. UWB1 cells were treated with or without nimbolide (1 μM for 48 hrs). The chromatin-bound fraction was isolated from these cells, and was subject to immunoblotting experiments using the indicated antibodies. b, Nimbolide treatment induces DNA damage response. HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The cell lysates were subject to immunoblotting experiments using the indicated antibodies. c, Nimbolide treatment induces the formation of cytosolic dsDNA and micronuclei. HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). DAPI (blue) was used to visualize the nuclei. Arrows indicate cytosolic dsDNA and micronuclei. Scale Bars, 10 μm. d, Nimbolide treatment induces the phosphorylation of TBK1. HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The level of pS172 TBK1 (p-TBK1, Green) was detected using the immunofluorescence assay. DAPI (blue) was used to visualize the nuclei. Scale Bars, 10 μm. e, Nimbolide treatment induces the phosphorylation of IRF3. HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The level of pS396 IRF3 (p-IRF3, Green) was detected using the immunofluorescence assay. DAPI (blue) was used to visualize the nuclei. Scale Bars, 10 μm. f, qRT-PCR analyses of IFN-β, CXCL10, or CCL5 in HeLa cells treated with or without nimbolide (1 μM for 48 hrs). g, Nimbolide treatment induces the expression of PD-L1. UWB1 cells were treated with or without nimbolide (1 μM for 48 hrs). PD-L1 expression was detected using the immunoblot assay. h, RNF114-KO abrogates the nimbolide-induced TBK1 phosphorylation. Control (RNF114-WT) and RNF114-KO HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The whole cell lysates were subject to immunoblot experiments using the indicated antibodies. i, PARP1-KO abrogates the nimbolide-induced TBK1 phosphorylation. Control (PARP1-WT) and PARP1-KO HeLa cells were treated with or without nimbolide (1 μM for 48 hrs). The whole cell lysates were subject to immunoblot experiments using the indicated antibodies.

    Techniques Used: Isolation, Western Blot, Immunofluorescence, Quantitative RT-PCR, Expressing

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    Cell Signaling Technology Inc anti irf3
    TLR9 activation upregulated PD-L1 expression by promoting STAT3 Tyr705 phosphorylation in HCC cells. ( A ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 5, 10, or 20μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( B ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, or 10μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( C ) PD-L1 protein expression after treatment with ODN1585 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, and 10 μM; times: 0, 6, 12, 24, and 36 hours in 5μM) in Hepa1-6 cells. PD-L1 protein levels were analyzed by Western blotting. ( D ) PD-L1 + tumor cells were detected by flow cytometry after TLR9 agonist (Hep3B and Huh7 cells with ODN2216; Hepa1-6 cells with ODN1585) treatment with indicated concentration. (values are mean ± SD, *p < 0.05, **p < 0.01, ***p<0.001, NS indicates no significance). ( E ) PD-L1 expression after TLR9 overexpression in Huh7 cells. PD-L1 expression levels were analyzed by immunofluorescence. ( F ) PD-L1 protein expression after TLR9 overexpression in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( G ) PD-L1 protein expression in TLR9 knockdown or TLR9 rescue Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( H and I ) mRNA levels of PD-L1 in Hep3B ( H ) and Huh7 ( I ) cells measured by qRT-PCR after stimulation with different concentrations of ODN2216. (values are mean ± SD, *p < 0.05, **p < 0.01, NS indicates no significance) ( J ) TLR9 overexpression-induced PD-L1 expression after MYC, JUN, IRF1, <t>IRF3,</t> STAT1 or STAT3 silencing. PD-L1 mRNA expression in Huh7 cells was analyzed after TLR9 overexpression alone or in the presence of MYC-, JUN-, IRF1-, IRF3-, STAT1- or STAT3-specific siRNA or siRNA-NC. (values are mean ± SD, ***p<0.001). (K) p-STAT3 (Tyr705) levels in Hep3B cells after treatment with different concentrations of ODN2216 (a TLR9 agonist; ODN2216: 0, 2.5, 5, or 10μM) analyzed by Western blotting. ( L ) p-STAT3 (Tyr705) levels after TLR9 overexpression in Huh7 cells analyzed by Western blotting. ( M ) TLR9 overexpression-induced p-STAT3 (Tyr705) levels after TLR9 inhibition. p-STAT3 (Tyr705) levels were analyzed after TLR9 overexpression alone or in the presence of the TLR9 antagonist chloroquine diphosphate. ( N ) p-STAT3-induced PD-L1 levels after STAT3 inhibition. PD-L1 levels were analyzed after TLR9 overexpression alone or in the presence of the STAT3-specific small molecular inhibitor BP-1-102.
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    Cell Signaling Technology Inc irf3
    Identification of RIG-I as the key sensor in type I IFN response in RPE. (a, b) RNA but not DNA could be sensed by RPE to induce type I IFN response, suggesting the lack of DNA sensors in unstimulated RPE. THP-1 and THP-1 cGAS KO cells were used as controls (a). Cells were treated with indicated inducers at 0.25 μ g/mL, with or without the Lipofectamine transfection reagent, and intracellular ISG15 was measured by ELISA 24 h after stimulation. Each bar represents biological replicates ( n = 3) and is indicated as mean ± SD. Note that ARPE-19 without transfection did not respond to most tested inducers except for poly(I:C). ∗ p < 0.05 compared with corresponded vehicle-treated groups. # p < 0.05 compared with same induces in parental cells. (c) Type I IFN response was induced via the <t>RIG-I–MAVS–IRF3</t> axis in ARPE-19 cells. ARPE-19 cells were cultured 10 d after reaching confluence for screening purposes. Different key nodes for the activation of the IFN pathway were knocked down using siRNAs (at least 3 different siRNAs for each gene). Cells were then treated with indicated nucleic acids, and the release of secreted IFN- β was measured by ELISA 24 h after stimulation. Similar results were observed in 3 independent experiments with different inducers (indicated in different lines) or different siRNAs (shown as different column); a single representative experiment is shown as an example. A heatmap was used to better visualize the percentage (%) change in the results. The efficiency of the mRNA knockdown was validated by qPCR and shown at the top in the form of heatmap. About ~70% inhibition was observed in most tested siRNAs.
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    Cell Signaling Technology Inc irf3 antibody
    cGAS-STING pathway was activated by arginine starvation. (A) Cells overexpressing cGAS-V5 were deprived for 72 h and stained with anti-V5 antibodies. Scale bars, 10 μm. (B) CWR22Rv1 cells were deprived for indicate timepoints, and endogenous <t>IRF3</t> was immunoprecipitated. IRF3 phosphorylation and TBK1 association were determined by immunoblots. Fold changes are listed below each blot. (C) IRF3 phosphorylation level was quantified and normalized with the IRF3. (n = 3, **p < 0.01). (D) RT-qPCR analysis of type I interferon expression in CWR22Rv1 cells starved for 72 h. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (E) IFNβ secretion by CWR22Rv1 cells after arginine starvation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (F) IFNβ promoter activity in CWR22Rv1 cells deprived for 72 h. (n = 3, *p < 0.05)
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    Cell Signaling Technology Inc rabbit irf3 antibody
    cGAS-STING pathway was activated by arginine starvation. (A) Cells overexpressing cGAS-V5 were deprived for 72 h and stained with anti-V5 antibodies. Scale bars, 10 μm. (B) CWR22Rv1 cells were deprived for indicate timepoints, and endogenous <t>IRF3</t> was immunoprecipitated. IRF3 phosphorylation and TBK1 association were determined by immunoblots. Fold changes are listed below each blot. (C) IRF3 phosphorylation level was quantified and normalized with the IRF3. (n = 3, **p < 0.01). (D) RT-qPCR analysis of type I interferon expression in CWR22Rv1 cells starved for 72 h. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (E) IFNβ secretion by CWR22Rv1 cells after arginine starvation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (F) IFNβ promoter activity in CWR22Rv1 cells deprived for 72 h. (n = 3, *p < 0.05)
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    Cell Signaling Technology Inc ifr3 11904s
    cGAS-STING pathway was activated by arginine starvation. (A) Cells overexpressing cGAS-V5 were deprived for 72 h and stained with anti-V5 antibodies. Scale bars, 10 μm. (B) CWR22Rv1 cells were deprived for indicate timepoints, and endogenous <t>IRF3</t> was immunoprecipitated. IRF3 phosphorylation and TBK1 association were determined by immunoblots. Fold changes are listed below each blot. (C) IRF3 phosphorylation level was quantified and normalized with the IRF3. (n = 3, **p < 0.01). (D) RT-qPCR analysis of type I interferon expression in CWR22Rv1 cells starved for 72 h. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (E) IFNβ secretion by CWR22Rv1 cells after arginine starvation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (F) IFNβ promoter activity in CWR22Rv1 cells deprived for 72 h. (n = 3, *p < 0.05)
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    Cell Signaling Technology Inc anti irf3 11904 antibodies
    (A) Human cGAS mutants used for functional analyses. Human cGAS is shown as a ribbon model, with the same coloring as in . Mutated residues are shown as red ball-and-stick models. (B) cGAS-induced phosphorylation of TBK1, <t>IRF3,</t> and STING. cGAS WT or mutants were expressed in HEK293T cells stably expressing human STING. Cell lysates were analyzed by western blotting, using the indicated antibodies. The asterisk indicates phosphorylated STING. (C) Reporter assays for IFN-β (left panel) and NF-κB (right panel). The cell lysates are the same as in (B), except for the co-expression with reporter plasmids, and were measured for luciferase activities. Luciferase activities are shown as mean ± s.d. (n = 3). (D) Induction of IFN-β and A20 by human cGAS and its mutants. The relative mRNA expression levels of IFN-β (left) and A20 (right) were analyzed by Real-Time PCR using total RNAs isolated from the cells shown in (B). Relative expression levels are shown as mean ± s.d. (n = 3). (E) Immunoblotting for cGAS-induced phosphorylation (left panel), reporter assays for IFN-β (upper panel) and NF-κB (lower panel), the same as (B) and (C), respectively. (F) Pull-down experiment between biotinylated-ISD and human cGAS mutants. Human cGAS mutants were expressed and purified from E. coli , and mixed with Streptavidin beads in the presence or absence of biotinylated-ISD. Bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE.
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    Cell Signaling Technology Inc rabbit monoclonal anti irf3 d6i4c
    a , Schematic diagram of the CTT of human STING (hSTING) and sequence logo of the CTT as indicated from 50 species. b , HeLa STING-knockout (KO) cells transfected with Flag-tagged STING ΔCΤΤ (Δ1–341), wild-type (WT) STING, STING E (E360A), STING LI (L364A/I365A) or STING ELI (E360A/L364A/I365A) were treated with diABZI for 2 h. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. c , WCL and extracted CCV fractions from HeLa STING KO cells reconstituted with Flag-tagged wild-type STING or STING LI (L364A/I365A) and treated or not with diABZI were analysed by western blot. CHC was used as a loading control. d , Glutathione sepharose pull-down assays of wild-type LBD-STING or LBD-STING ELI by glutathione S -transferase (GST)-tagged AP-1 core with or without ARF1. e , HeLa STING cells transfected with NC siRNA or siRNAs against TBK1 or <t>IRF3</t> were treated with or without diABZI. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. GAPDH was used as a loading control. f , HeLa wild-type cells, HeLa TBK1 KO cells and HeLa IRF3 KO cells stimulated with diABZI for 0, 1, 2 or 4 h were analysed by western blot. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition. Vinculin was used as a loading control. g , Bio-layer interferometry binding studies of LBD-STING (top) or TBK1-phosphorylated LBD-STING (pSTING) (bottom) with AP-1 ΔμCTD. The right graphs show the binding affinity of STING (top) and pSTING (bottom). One representative example of at least three ( b , d , f ) or two ( c , e , g ) independent experiments is shown.
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    TLR9 activation upregulated PD-L1 expression by promoting STAT3 Tyr705 phosphorylation in HCC cells. ( A ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 5, 10, or 20μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( B ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, or 10μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( C ) PD-L1 protein expression after treatment with ODN1585 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, and 10 μM; times: 0, 6, 12, 24, and 36 hours in 5μM) in Hepa1-6 cells. PD-L1 protein levels were analyzed by Western blotting. ( D ) PD-L1 + tumor cells were detected by flow cytometry after TLR9 agonist (Hep3B and Huh7 cells with ODN2216; Hepa1-6 cells with ODN1585) treatment with indicated concentration. (values are mean ± SD, *p < 0.05, **p < 0.01, ***p<0.001, NS indicates no significance). ( E ) PD-L1 expression after TLR9 overexpression in Huh7 cells. PD-L1 expression levels were analyzed by immunofluorescence. ( F ) PD-L1 protein expression after TLR9 overexpression in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( G ) PD-L1 protein expression in TLR9 knockdown or TLR9 rescue Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( H and I ) mRNA levels of PD-L1 in Hep3B ( H ) and Huh7 ( I ) cells measured by qRT-PCR after stimulation with different concentrations of ODN2216. (values are mean ± SD, *p < 0.05, **p < 0.01, NS indicates no significance) ( J ) TLR9 overexpression-induced PD-L1 expression after MYC, JUN, IRF1, IRF3, STAT1 or STAT3 silencing. PD-L1 mRNA expression in Huh7 cells was analyzed after TLR9 overexpression alone or in the presence of MYC-, JUN-, IRF1-, IRF3-, STAT1- or STAT3-specific siRNA or siRNA-NC. (values are mean ± SD, ***p<0.001). (K) p-STAT3 (Tyr705) levels in Hep3B cells after treatment with different concentrations of ODN2216 (a TLR9 agonist; ODN2216: 0, 2.5, 5, or 10μM) analyzed by Western blotting. ( L ) p-STAT3 (Tyr705) levels after TLR9 overexpression in Huh7 cells analyzed by Western blotting. ( M ) TLR9 overexpression-induced p-STAT3 (Tyr705) levels after TLR9 inhibition. p-STAT3 (Tyr705) levels were analyzed after TLR9 overexpression alone or in the presence of the TLR9 antagonist chloroquine diphosphate. ( N ) p-STAT3-induced PD-L1 levels after STAT3 inhibition. PD-L1 levels were analyzed after TLR9 overexpression alone or in the presence of the STAT3-specific small molecular inhibitor BP-1-102.

    Journal: Theranostics

    Article Title: Hepatoma cell-intrinsic TLR9 activation induces immune escape through PD-L1 upregulation in hepatocellular carcinoma

    doi: 10.7150/thno.44417

    Figure Lengend Snippet: TLR9 activation upregulated PD-L1 expression by promoting STAT3 Tyr705 phosphorylation in HCC cells. ( A ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 5, 10, or 20μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( B ) PD-L1 protein expression after treatment with ODN2216 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, or 10μM; times: 0, 6, 12, 24, and 36 hours in 10 μM) in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( C ) PD-L1 protein expression after treatment with ODN1585 with different concentrations or the indicated times (different concentrations: 0, 2.5, 5, and 10 μM; times: 0, 6, 12, 24, and 36 hours in 5μM) in Hepa1-6 cells. PD-L1 protein levels were analyzed by Western blotting. ( D ) PD-L1 + tumor cells were detected by flow cytometry after TLR9 agonist (Hep3B and Huh7 cells with ODN2216; Hepa1-6 cells with ODN1585) treatment with indicated concentration. (values are mean ± SD, *p < 0.05, **p < 0.01, ***p<0.001, NS indicates no significance). ( E ) PD-L1 expression after TLR9 overexpression in Huh7 cells. PD-L1 expression levels were analyzed by immunofluorescence. ( F ) PD-L1 protein expression after TLR9 overexpression in Huh7 cells. PD-L1 protein levels were analyzed by Western blotting. ( G ) PD-L1 protein expression in TLR9 knockdown or TLR9 rescue Hep3B cells. PD-L1 protein levels were analyzed by Western blotting. ( H and I ) mRNA levels of PD-L1 in Hep3B ( H ) and Huh7 ( I ) cells measured by qRT-PCR after stimulation with different concentrations of ODN2216. (values are mean ± SD, *p < 0.05, **p < 0.01, NS indicates no significance) ( J ) TLR9 overexpression-induced PD-L1 expression after MYC, JUN, IRF1, IRF3, STAT1 or STAT3 silencing. PD-L1 mRNA expression in Huh7 cells was analyzed after TLR9 overexpression alone or in the presence of MYC-, JUN-, IRF1-, IRF3-, STAT1- or STAT3-specific siRNA or siRNA-NC. (values are mean ± SD, ***p<0.001). (K) p-STAT3 (Tyr705) levels in Hep3B cells after treatment with different concentrations of ODN2216 (a TLR9 agonist; ODN2216: 0, 2.5, 5, or 10μM) analyzed by Western blotting. ( L ) p-STAT3 (Tyr705) levels after TLR9 overexpression in Huh7 cells analyzed by Western blotting. ( M ) TLR9 overexpression-induced p-STAT3 (Tyr705) levels after TLR9 inhibition. p-STAT3 (Tyr705) levels were analyzed after TLR9 overexpression alone or in the presence of the TLR9 antagonist chloroquine diphosphate. ( N ) p-STAT3-induced PD-L1 levels after STAT3 inhibition. PD-L1 levels were analyzed after TLR9 overexpression alone or in the presence of the STAT3-specific small molecular inhibitor BP-1-102.

    Article Snippet: The antibodies listed below were used in Western blotting, immunohistochemical and flow cytometry analyses: anti-TLR9 (ab37154, Abcam, Cambridge, UK; NBP2-24729, Novus Biologicals, Minneapolis, USA), anti-PARP1 (#9532; Cell Signaling Technology, Danvers, MA, USA; ab227244, Abcam), anti-PAR (#83732; Cell Signaling Technology, Danvers, MA, USA), anti-Ubiquitin (#3936; Cell Signaling Technology, Danvers, MA, USA); anti-STAT3 (#9139; Cell Signaling Technology, Danvers, MA, USA), anti-p-STAT3 (Tyr705) (#9145; Cell Signaling Technology, Danvers, MA, USA); anti-PD-L1 (#13684T, Cell Signaling Technology, Danvers, MA, USA; 329702, BioLegend, San Diego, CA, USA; ab205921, Abcam, Cambridge, UK), anti-Jak2 (#3230; Cell Signaling Technology, Danvers, MA, USA), anti-p-Jak2 (#3774; Cell Signaling Technology, Danvers, MA, USA), anti-STAT1(#14994, Cell Signaling Technology, Danvers, MA, USA), anti-JUN(#9165; Cell Signaling Technology, Danvers, MA, USA), anti-IRF1(#8478; Cell Signaling Technology, Danvers, MA, USA), anti-IRF3(#11904; Cell Signaling Technology, Danvers, MA, USA); anti-Myc (Sigma-Aldrich, St. Louis, MO, USA), anti-granzyme B (ab4059; Abcam), anti-CD8 (ab22378; Abcam; 560776; BD Biosciences), anti-JNK (AF6318; Affinity Biosciences, USA), anti-p-JNK (AF3318; Affinity Biosciences, USA), anti-ERK (AF0155; Affinity Biosciences, USA), anti-p-ERK (AF1015; Affinity Biosciences, USA), anti-p-p38 (AF4001; Affinity Biosciences, USA), anti-p38 (AF6456; Affinity Biosciences, USA), anti-PARG (#27808; Proteintech, Wuhan), anti-RNF146 (#ARP43340; Aviva Systems Biology Co., Ltd. USA; #bs-11669R; Bioss Antibodies Inc. USA), anti-CD4 (550954; BD Biosciences), anti-PD-1 (551892; BD Biosciences), anti-CD11b (557395; BD Biosciences), anti-NK1.1 (557391; BD Biosciences), anti-CD25 (553071; BD Biosciences), anti-Foxp3 (560402; BD Biosciences), anti-F4/80 (565612;BD Bioscience), anti-CD11c (553800; BD Biosciences), anti-CD317 (566431; BD Biosciences), PDD-00017273 (#5952; Tocris Bioscience, USA), ODN2216 (#tlrl-2216; Invivogen, USA), ODN2243(ODN2216 Control, #tlrl-2243; Invivogen, USA) and SP600125 (#T3109), U0126(#T6223), SB203580(#T1764), BP-1-102 (#T3708), Chloroquine diphosphate (#T0194) were purchased from Topscience (Topscience Co., Ltd. Shanghai).

    Techniques: Activation Assay, Expressing, Western Blot, Flow Cytometry, Concentration Assay, Over Expression, Immunofluorescence, Quantitative RT-PCR, Inhibition

    Identification of RIG-I as the key sensor in type I IFN response in RPE. (a, b) RNA but not DNA could be sensed by RPE to induce type I IFN response, suggesting the lack of DNA sensors in unstimulated RPE. THP-1 and THP-1 cGAS KO cells were used as controls (a). Cells were treated with indicated inducers at 0.25 μ g/mL, with or without the Lipofectamine transfection reagent, and intracellular ISG15 was measured by ELISA 24 h after stimulation. Each bar represents biological replicates ( n = 3) and is indicated as mean ± SD. Note that ARPE-19 without transfection did not respond to most tested inducers except for poly(I:C). ∗ p < 0.05 compared with corresponded vehicle-treated groups. # p < 0.05 compared with same induces in parental cells. (c) Type I IFN response was induced via the RIG-I–MAVS–IRF3 axis in ARPE-19 cells. ARPE-19 cells were cultured 10 d after reaching confluence for screening purposes. Different key nodes for the activation of the IFN pathway were knocked down using siRNAs (at least 3 different siRNAs for each gene). Cells were then treated with indicated nucleic acids, and the release of secreted IFN- β was measured by ELISA 24 h after stimulation. Similar results were observed in 3 independent experiments with different inducers (indicated in different lines) or different siRNAs (shown as different column); a single representative experiment is shown as an example. A heatmap was used to better visualize the percentage (%) change in the results. The efficiency of the mRNA knockdown was validated by qPCR and shown at the top in the form of heatmap. About ~70% inhibition was observed in most tested siRNAs.

    Journal: Journal of Immunology Research

    Article Title: Mechanism of Nucleic Acid Sensing in Retinal Pigment Epithelium (RPE): RIG-I Mediates Type I Interferon Response in Human RPE

    doi: 10.1155/2021/9975628

    Figure Lengend Snippet: Identification of RIG-I as the key sensor in type I IFN response in RPE. (a, b) RNA but not DNA could be sensed by RPE to induce type I IFN response, suggesting the lack of DNA sensors in unstimulated RPE. THP-1 and THP-1 cGAS KO cells were used as controls (a). Cells were treated with indicated inducers at 0.25 μ g/mL, with or without the Lipofectamine transfection reagent, and intracellular ISG15 was measured by ELISA 24 h after stimulation. Each bar represents biological replicates ( n = 3) and is indicated as mean ± SD. Note that ARPE-19 without transfection did not respond to most tested inducers except for poly(I:C). ∗ p < 0.05 compared with corresponded vehicle-treated groups. # p < 0.05 compared with same induces in parental cells. (c) Type I IFN response was induced via the RIG-I–MAVS–IRF3 axis in ARPE-19 cells. ARPE-19 cells were cultured 10 d after reaching confluence for screening purposes. Different key nodes for the activation of the IFN pathway were knocked down using siRNAs (at least 3 different siRNAs for each gene). Cells were then treated with indicated nucleic acids, and the release of secreted IFN- β was measured by ELISA 24 h after stimulation. Similar results were observed in 3 independent experiments with different inducers (indicated in different lines) or different siRNAs (shown as different column); a single representative experiment is shown as an example. A heatmap was used to better visualize the percentage (%) change in the results. The efficiency of the mRNA knockdown was validated by qPCR and shown at the top in the form of heatmap. About ~70% inhibition was observed in most tested siRNAs.

    Article Snippet: Following three washes with TBST, the membrane was incubated overnight with primary antibodies purchased from Cell Signaling Technology, including cGAS (CST#15102), IRF3 (CST#11904), RIG-I (CST#3743), Histone H3 (CST#4499), β -actin (CST#5125), and GAPDH (CST#8884), at 1 : 1000 dilution.

    Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, Inhibition

    cGAS-STING pathway was activated by arginine starvation. (A) Cells overexpressing cGAS-V5 were deprived for 72 h and stained with anti-V5 antibodies. Scale bars, 10 μm. (B) CWR22Rv1 cells were deprived for indicate timepoints, and endogenous IRF3 was immunoprecipitated. IRF3 phosphorylation and TBK1 association were determined by immunoblots. Fold changes are listed below each blot. (C) IRF3 phosphorylation level was quantified and normalized with the IRF3. (n = 3, **p < 0.01). (D) RT-qPCR analysis of type I interferon expression in CWR22Rv1 cells starved for 72 h. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (E) IFNβ secretion by CWR22Rv1 cells after arginine starvation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (F) IFNβ promoter activity in CWR22Rv1 cells deprived for 72 h. (n = 3, *p < 0.05)

    Journal: Theranostics

    Article Title: Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes

    doi: 10.7150/thno.54695

    Figure Lengend Snippet: cGAS-STING pathway was activated by arginine starvation. (A) Cells overexpressing cGAS-V5 were deprived for 72 h and stained with anti-V5 antibodies. Scale bars, 10 μm. (B) CWR22Rv1 cells were deprived for indicate timepoints, and endogenous IRF3 was immunoprecipitated. IRF3 phosphorylation and TBK1 association were determined by immunoblots. Fold changes are listed below each blot. (C) IRF3 phosphorylation level was quantified and normalized with the IRF3. (n = 3, **p < 0.01). (D) RT-qPCR analysis of type I interferon expression in CWR22Rv1 cells starved for 72 h. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (E) IFNβ secretion by CWR22Rv1 cells after arginine starvation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (F) IFNβ promoter activity in CWR22Rv1 cells deprived for 72 h. (n = 3, *p < 0.05)

    Article Snippet: IRF3 was immunoprecipitated with the IRF3 antibody (Cell Signaling, 11904) or control rabbit IgG (Millipore, pp64), and the precipitated immune complex was detected with IRF3, p-IRF3 (S396), TBK1 and p-TBK1 (S172) antibodies.

    Techniques: Staining, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Expressing, Activity Assay

    Translocation of the cGAS downstream effectors. (A) Nuclear translocation of p65 was determined by subcellular fractionation in arginine-deprived CWR22Rv1 cells. (B) Nuclear p65 was quantified and normalized to lamin A/C. (n = 3, ***p < 0.001) (C) Immunostaining of p65 in CWR22Rv1 cells. Scale bars, 10 μm. (D) ChIP-qPCR analysis of IRF3 binding on the promoter regions of IFNA14 and IFNB1 in CWR22Rv1. (n = 3, *p < 0.05) (E) CWR22Rv1 cells overexpressing STING-V5 were subjected to arginine deprivation and stained with V5, calnexin and RCAS1 antibodies. Scale bars, 10 μm.

    Journal: Theranostics

    Article Title: Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes

    doi: 10.7150/thno.54695

    Figure Lengend Snippet: Translocation of the cGAS downstream effectors. (A) Nuclear translocation of p65 was determined by subcellular fractionation in arginine-deprived CWR22Rv1 cells. (B) Nuclear p65 was quantified and normalized to lamin A/C. (n = 3, ***p < 0.001) (C) Immunostaining of p65 in CWR22Rv1 cells. Scale bars, 10 μm. (D) ChIP-qPCR analysis of IRF3 binding on the promoter regions of IFNA14 and IFNB1 in CWR22Rv1. (n = 3, *p < 0.05) (E) CWR22Rv1 cells overexpressing STING-V5 were subjected to arginine deprivation and stained with V5, calnexin and RCAS1 antibodies. Scale bars, 10 μm.

    Article Snippet: IRF3 was immunoprecipitated with the IRF3 antibody (Cell Signaling, 11904) or control rabbit IgG (Millipore, pp64), and the precipitated immune complex was detected with IRF3, p-IRF3 (S396), TBK1 and p-TBK1 (S172) antibodies.

    Techniques: Translocation Assay, Fractionation, Immunostaining, Binding Assay, Staining

    (A) Human cGAS mutants used for functional analyses. Human cGAS is shown as a ribbon model, with the same coloring as in . Mutated residues are shown as red ball-and-stick models. (B) cGAS-induced phosphorylation of TBK1, IRF3, and STING. cGAS WT or mutants were expressed in HEK293T cells stably expressing human STING. Cell lysates were analyzed by western blotting, using the indicated antibodies. The asterisk indicates phosphorylated STING. (C) Reporter assays for IFN-β (left panel) and NF-κB (right panel). The cell lysates are the same as in (B), except for the co-expression with reporter plasmids, and were measured for luciferase activities. Luciferase activities are shown as mean ± s.d. (n = 3). (D) Induction of IFN-β and A20 by human cGAS and its mutants. The relative mRNA expression levels of IFN-β (left) and A20 (right) were analyzed by Real-Time PCR using total RNAs isolated from the cells shown in (B). Relative expression levels are shown as mean ± s.d. (n = 3). (E) Immunoblotting for cGAS-induced phosphorylation (left panel), reporter assays for IFN-β (upper panel) and NF-κB (lower panel), the same as (B) and (C), respectively. (F) Pull-down experiment between biotinylated-ISD and human cGAS mutants. Human cGAS mutants were expressed and purified from E. coli , and mixed with Streptavidin beads in the presence or absence of biotinylated-ISD. Bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE.

    Journal: PLoS ONE

    Article Title: Structural and Functional Analyses of DNA-Sensing and Immune Activation by Human cGAS

    doi: 10.1371/journal.pone.0076983

    Figure Lengend Snippet: (A) Human cGAS mutants used for functional analyses. Human cGAS is shown as a ribbon model, with the same coloring as in . Mutated residues are shown as red ball-and-stick models. (B) cGAS-induced phosphorylation of TBK1, IRF3, and STING. cGAS WT or mutants were expressed in HEK293T cells stably expressing human STING. Cell lysates were analyzed by western blotting, using the indicated antibodies. The asterisk indicates phosphorylated STING. (C) Reporter assays for IFN-β (left panel) and NF-κB (right panel). The cell lysates are the same as in (B), except for the co-expression with reporter plasmids, and were measured for luciferase activities. Luciferase activities are shown as mean ± s.d. (n = 3). (D) Induction of IFN-β and A20 by human cGAS and its mutants. The relative mRNA expression levels of IFN-β (left) and A20 (right) were analyzed by Real-Time PCR using total RNAs isolated from the cells shown in (B). Relative expression levels are shown as mean ± s.d. (n = 3). (E) Immunoblotting for cGAS-induced phosphorylation (left panel), reporter assays for IFN-β (upper panel) and NF-κB (lower panel), the same as (B) and (C), respectively. (F) Pull-down experiment between biotinylated-ISD and human cGAS mutants. Human cGAS mutants were expressed and purified from E. coli , and mixed with Streptavidin beads in the presence or absence of biotinylated-ISD. Bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE.

    Article Snippet: Anti-STING (#3337), anti-phospho-TBK1 (#5483), anti-TBK1 (#3504), anti-phospho-IRF3 (#4947), and anti-IRF3 (#11904) antibodies were purchased from Cell Signaling.

    Techniques: Functional Assay, Stable Transfection, Expressing, Western Blot, Luciferase, Real-time Polymerase Chain Reaction, Isolation, Purification, SDS Page

    a , Schematic diagram of the CTT of human STING (hSTING) and sequence logo of the CTT as indicated from 50 species. b , HeLa STING-knockout (KO) cells transfected with Flag-tagged STING ΔCΤΤ (Δ1–341), wild-type (WT) STING, STING E (E360A), STING LI (L364A/I365A) or STING ELI (E360A/L364A/I365A) were treated with diABZI for 2 h. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. c , WCL and extracted CCV fractions from HeLa STING KO cells reconstituted with Flag-tagged wild-type STING or STING LI (L364A/I365A) and treated or not with diABZI were analysed by western blot. CHC was used as a loading control. d , Glutathione sepharose pull-down assays of wild-type LBD-STING or LBD-STING ELI by glutathione S -transferase (GST)-tagged AP-1 core with or without ARF1. e , HeLa STING cells transfected with NC siRNA or siRNAs against TBK1 or IRF3 were treated with or without diABZI. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. GAPDH was used as a loading control. f , HeLa wild-type cells, HeLa TBK1 KO cells and HeLa IRF3 KO cells stimulated with diABZI for 0, 1, 2 or 4 h were analysed by western blot. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition. Vinculin was used as a loading control. g , Bio-layer interferometry binding studies of LBD-STING (top) or TBK1-phosphorylated LBD-STING (pSTING) (bottom) with AP-1 ΔμCTD. The right graphs show the binding affinity of STING (top) and pSTING (bottom). One representative example of at least three ( b , d , f ) or two ( c , e , g ) independent experiments is shown.

    Journal: Nature

    Article Title: Clathrin-associated AP-1 controls termination of STING signalling

    doi: 10.1038/s41586-022-05354-0

    Figure Lengend Snippet: a , Schematic diagram of the CTT of human STING (hSTING) and sequence logo of the CTT as indicated from 50 species. b , HeLa STING-knockout (KO) cells transfected with Flag-tagged STING ΔCΤΤ (Δ1–341), wild-type (WT) STING, STING E (E360A), STING LI (L364A/I365A) or STING ELI (E360A/L364A/I365A) were treated with diABZI for 2 h. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. c , WCL and extracted CCV fractions from HeLa STING KO cells reconstituted with Flag-tagged wild-type STING or STING LI (L364A/I365A) and treated or not with diABZI were analysed by western blot. CHC was used as a loading control. d , Glutathione sepharose pull-down assays of wild-type LBD-STING or LBD-STING ELI by glutathione S -transferase (GST)-tagged AP-1 core with or without ARF1. e , HeLa STING cells transfected with NC siRNA or siRNAs against TBK1 or IRF3 were treated with or without diABZI. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. GAPDH was used as a loading control. f , HeLa wild-type cells, HeLa TBK1 KO cells and HeLa IRF3 KO cells stimulated with diABZI for 0, 1, 2 or 4 h were analysed by western blot. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition. Vinculin was used as a loading control. g , Bio-layer interferometry binding studies of LBD-STING (top) or TBK1-phosphorylated LBD-STING (pSTING) (bottom) with AP-1 ΔμCTD. The right graphs show the binding affinity of STING (top) and pSTING (bottom). One representative example of at least three ( b , d , f ) or two ( c , e , g ) independent experiments is shown.

    Article Snippet: Primary antibodies used: mouse monoclonal anti-vinculin (hVIN-1) (Sigma-Aldrich, V9264, immunoblot 1:5,000), rabbit monoclonal anti-GAPDH (14C10) (Cell Signaling Technology, 2118, immunoblot 1:3,000), mouse monoclonal anti-Flag (M2) (Sigma-Aldrich, F1804, immunoblot 1:3,000), rabbit monoclonal anti-human phospho-STING (Ser366) (D7C3S) (Cell Signaling Technology, 19781, immunoblot 1:3,000), rabbit monoclonal anti-phospho-TBK1/NAK (Ser172) (D52C2) (Cell Signaling Technology, 5483, immunoblot 1:1,000), rabbit monoclonal anti-phospho-IRF3 (Ser386) (EPR2346) (Abcam, ab76493, immunoblot 1:1,000), rabbit monoclonal anti-TBK1/NAK (D1B4) (Cell Signaling Technology, 3504, immunoblot 1:1,000), rabbit polyclonal anti-TMEM173/STING (Proteintech, 19851-1-AP, immunoblot 1:1,000), rabbit monoclonal anti-IRF3 (D6I4C) (Cell Signaling Technology, 11904, immunoblot 1:1,000), rabbit monoclonal anti-clathrin heavy chain (P1663) (Cell Signaling Technology, 2410, immunoblot 1:500), mouse anti-clathrin heavy chain monoclonal antibody (X22) (Thermo Fisher Scientific, MA1-065, immunofluorescence (IF) 1:100), rabbit polyclonal anti-AP1S1 (Thermo Fisher Scientific, PA5-63913, immunoblot 1:1,000), rabbit polyclonal anti-AP1G1 (Thermo Fisher Scientific, PA5-65290, immunoblot 1:1,000), rabbit polyclonal anti-AP1B1 (Sigma-Aldrich, HPA065226, immunoblot 1:1,000), rabbit polyclonal anti-AP1M1 (Proteintech, 12112-1-AP, immunoblot 1:1,000), mouse monoclonal anti-HSV-1 ICP0 (11060) (Santa Cruz, sc-53090, immunoblot 1:500), mouse monoclonal anti-HA.11 epitope tag (16B12) (Biolegend, MMS-101R, immunoblot 1:2,000). mouse monoclonal γ-adaptin (AP1G1) (100/3) (Sigma-Aldrich, A4200, IF 1:100), mouse monoclonal EEA1 (E9Q6G) (Cell Signaling, 48453, IF 1:100), mouse monoclonal LAMP1 (H4A3) (Abcam, ab25630, IF 1:100), rabbit monoclonal anti-human phospho-STING (Ser366) (D8K6H) (Cell Signaling Technology, 40818, IF 1:100, STED 1:50), mouse monoclonal RAB7 (E9O7E) (Cell Signaling Technology, 95746, IF 1:100) and sheep polyclonal human TGN46 (Bio-Rad, AHP500G, IF 1:200).

    Techniques: Sequencing, Knock-Out, Transfection, Immunoprecipitation, Western Blot, Binding Assay