tbk1 nak d1b4 rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc tbk1 nak d1b4 rabbit mab
    Tbk1 Nak D1b4 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tbk1 nak d1b4 rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc tbk1 nak d1b4 rabbit mab
    Tbk1 Nak D1b4 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti tbk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti tbk1
    Anti Tbk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tbk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc tbk1
    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 <t>TBK1</t> 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)
    Tbk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, 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

    tbk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc tbk1
    cGAS Regulates Acute Intestinal Inflammation. (A) Experimental design. WT and Cgas -/- mice treated with water or 3% DSS in drinking water for 7 days and followed for normal water for 4 days. (B) Weight loss in DSS-exposed mice. (C) Disease activity index (DAI) of DSS-exposed mice. (D) Kaplan-Meier survival curve for 11 days after DSS treatment. (E) Length of the colons of WT or Cgas -/- mice treated with DSS or control at day 9. (F) Myeloperoxidase (MPO) activity in colon homogenates at day 9. (G) Colonic Cgas , Sting, Ifit-1, and Ifn-β transcripts determined in WT or Cgas -/- mice undergoing DSS-colitis at day 9. (H) Western blot analyses of colons at day 9. Lysates were probed against cGAS, STING, p65, <t>p-TBK1,</t> TBK1, and β-actin. (I) Inflammatory cytokine expression was quantified in whole colon tissues from WT or Cgas -/- mice at day 9 by ELISA. Data are presented as the mean ± s.d. n = 5-7 mice/group from three independent experiments; statistical significance determined by one-way ANOVA (Turkey's post hoc) or two-way ANOVA. ns , no significant. See also .
    Tbk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Microbial and genetic-based framework identifies drug targets in inflammatory bowel disease"

    Article Title: Microbial and genetic-based framework identifies drug targets in inflammatory bowel disease

    Journal: Theranostics

    doi: 10.7150/thno.59196

    cGAS Regulates Acute Intestinal Inflammation. (A) Experimental design. WT and Cgas -/- mice treated with water or 3% DSS in drinking water for 7 days and followed for normal water for 4 days. (B) Weight loss in DSS-exposed mice. (C) Disease activity index (DAI) of DSS-exposed mice. (D) Kaplan-Meier survival curve for 11 days after DSS treatment. (E) Length of the colons of WT or Cgas -/- mice treated with DSS or control at day 9. (F) Myeloperoxidase (MPO) activity in colon homogenates at day 9. (G) Colonic Cgas , Sting, Ifit-1, and Ifn-β transcripts determined in WT or Cgas -/- mice undergoing DSS-colitis at day 9. (H) Western blot analyses of colons at day 9. Lysates were probed against cGAS, STING, p65, p-TBK1, TBK1, and β-actin. (I) Inflammatory cytokine expression was quantified in whole colon tissues from WT or Cgas -/- mice at day 9 by ELISA. Data are presented as the mean ± s.d. n = 5-7 mice/group from three independent experiments; statistical significance determined by one-way ANOVA (Turkey's post hoc) or two-way ANOVA. ns , no significant. See also .
    Figure Legend Snippet: cGAS Regulates Acute Intestinal Inflammation. (A) Experimental design. WT and Cgas -/- mice treated with water or 3% DSS in drinking water for 7 days and followed for normal water for 4 days. (B) Weight loss in DSS-exposed mice. (C) Disease activity index (DAI) of DSS-exposed mice. (D) Kaplan-Meier survival curve for 11 days after DSS treatment. (E) Length of the colons of WT or Cgas -/- mice treated with DSS or control at day 9. (F) Myeloperoxidase (MPO) activity in colon homogenates at day 9. (G) Colonic Cgas , Sting, Ifit-1, and Ifn-β transcripts determined in WT or Cgas -/- mice undergoing DSS-colitis at day 9. (H) Western blot analyses of colons at day 9. Lysates were probed against cGAS, STING, p65, p-TBK1, TBK1, and β-actin. (I) Inflammatory cytokine expression was quantified in whole colon tissues from WT or Cgas -/- mice at day 9 by ELISA. Data are presented as the mean ± s.d. n = 5-7 mice/group from three independent experiments; statistical significance determined by one-way ANOVA (Turkey's post hoc) or two-way ANOVA. ns , no significant. See also .

    Techniques Used: Activity Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

    tbk1 nak  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc tbk1 nak
    Transcriptional regulation of T H 1 and T H 9 cell differentiation following STING activation. (A) Heatmap representing Ifng , Tbx21 , Irf1 , Gata3 , Il9 , Irf4 , Irf8 , Batf , Rorc , and Foxp3 mRNA expression (Z-score) from naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 72 hours. (B–D) WT naive CD4 T cells stimulated with cGAMP or control for 4(B) or 6(C, D) hours. (B) STING, p-STING, <t>TBK1</t> and p-TBK1 protein levels. (C) Cyt and Nuc localization of IRF3, p65 NF-κB and their phosphorylated forms. (D) Heatmap representing Ifna , Ifnb1 , Il6 , Tnfa , Ifit1 , Ifit2 , Mx2 and Cxcl10 mRNA expression (Log2FC) between CD4 T cells stimulated with cGAMP and control. Mean of replicates from three independent experiments. P values (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) determined by two-way analysis of variance (A) or unpaired t test (D). (E, F) RNA sequencing analysis from WT naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 16 hours. Biological replicates from three independent experiments. (E) Gene set enrichment analysis comparing expression in cGAMP-stimulated cells to control cells. Enrichment plot and score, Nom. P value and FDR q value shown for the three gene sets. (F) Heatmaps illustrating the hierarchical clustering of expression levels (rld values) of ISGs. cGAMP, 2′3′-cyclic guanosine monophosphate–adenosine monophosphate; Cyt, cytosolic; FDR, false discovery rate; ISG, interferon-stimulated gene; Nom, nominal; Nuc, nuclear; STING, stimulator of interferon genes; WT, wild type.
    Tbk1 Nak, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "CD4 T cell-intrinsic STING signaling controls the differentiation and effector functions of T H 1 and T H 9 cells"

    Article Title: CD4 T cell-intrinsic STING signaling controls the differentiation and effector functions of T H 1 and T H 9 cells

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1136/jitc-2021-003459

    Transcriptional regulation of T H 1 and T H 9 cell differentiation following STING activation. (A) Heatmap representing Ifng , Tbx21 , Irf1 , Gata3 , Il9 , Irf4 , Irf8 , Batf , Rorc , and Foxp3 mRNA expression (Z-score) from naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 72 hours. (B–D) WT naive CD4 T cells stimulated with cGAMP or control for 4(B) or 6(C, D) hours. (B) STING, p-STING, TBK1 and p-TBK1 protein levels. (C) Cyt and Nuc localization of IRF3, p65 NF-κB and their phosphorylated forms. (D) Heatmap representing Ifna , Ifnb1 , Il6 , Tnfa , Ifit1 , Ifit2 , Mx2 and Cxcl10 mRNA expression (Log2FC) between CD4 T cells stimulated with cGAMP and control. Mean of replicates from three independent experiments. P values (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) determined by two-way analysis of variance (A) or unpaired t test (D). (E, F) RNA sequencing analysis from WT naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 16 hours. Biological replicates from three independent experiments. (E) Gene set enrichment analysis comparing expression in cGAMP-stimulated cells to control cells. Enrichment plot and score, Nom. P value and FDR q value shown for the three gene sets. (F) Heatmaps illustrating the hierarchical clustering of expression levels (rld values) of ISGs. cGAMP, 2′3′-cyclic guanosine monophosphate–adenosine monophosphate; Cyt, cytosolic; FDR, false discovery rate; ISG, interferon-stimulated gene; Nom, nominal; Nuc, nuclear; STING, stimulator of interferon genes; WT, wild type.
    Figure Legend Snippet: Transcriptional regulation of T H 1 and T H 9 cell differentiation following STING activation. (A) Heatmap representing Ifng , Tbx21 , Irf1 , Gata3 , Il9 , Irf4 , Irf8 , Batf , Rorc , and Foxp3 mRNA expression (Z-score) from naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 72 hours. (B–D) WT naive CD4 T cells stimulated with cGAMP or control for 4(B) or 6(C, D) hours. (B) STING, p-STING, TBK1 and p-TBK1 protein levels. (C) Cyt and Nuc localization of IRF3, p65 NF-κB and their phosphorylated forms. (D) Heatmap representing Ifna , Ifnb1 , Il6 , Tnfa , Ifit1 , Ifit2 , Mx2 and Cxcl10 mRNA expression (Log2FC) between CD4 T cells stimulated with cGAMP and control. Mean of replicates from three independent experiments. P values (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) determined by two-way analysis of variance (A) or unpaired t test (D). (E, F) RNA sequencing analysis from WT naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 16 hours. Biological replicates from three independent experiments. (E) Gene set enrichment analysis comparing expression in cGAMP-stimulated cells to control cells. Enrichment plot and score, Nom. P value and FDR q value shown for the three gene sets. (F) Heatmaps illustrating the hierarchical clustering of expression levels (rld values) of ISGs. cGAMP, 2′3′-cyclic guanosine monophosphate–adenosine monophosphate; Cyt, cytosolic; FDR, false discovery rate; ISG, interferon-stimulated gene; Nom, nominal; Nuc, nuclear; STING, stimulator of interferon genes; WT, wild type.

    Techniques Used: Cell Differentiation, Activation Assay, Expressing, RNA Sequencing Assay

    anti tbk1 3504  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti tbk1 3504
    IKKε directly phosphorylates kindlin-2 at S159. (A) Slot blot analysis of the anti-phospho-kindlin-2-S159 antibody. The antibody was probed against an un-modified kindlin-2 peptide and phosphorylated peptides. (B) HCT116 cells were transfected with the indicated plasmids and whole cell extracts were immunoprecipitated (IP) with FLAG-conjugated M2 beads and then analyzed by immunoblotting (IB). (C)HCT116 cells were treated with the inhibitor IKK-3 at the indicated concentration for 24h and analyzed by immunoblotting. (D and E) HCT116 cells were transfected with the indicated siRNAs targeting IKKα, IKKβ, IKKε or <t>TBK1.</t> Cell lysates were analyzed by immunoblotting. (F) HCT116 cells were transfected with FLAG-tagged kindlin-2 and GFP-tagged IKKε for 48 h. Total cell lysates were immunoprecipitated using FLAG-conjugated M2 beads and analyzed by immunoblotting. (G) IKKε was immunoprecipitated using anti-IKKε antibody or the control antibody IgG from lysates of HCT116 cells. (H) HCT116 cells were transfected with the indicated plasmids and the whole cell extracts were prepared and analyzed by Co-IP assay flowed by immunoblotting with the indicated antibodies. (I) GST-tagged wild-type kindlin-2 or kindlin-2 (S159A) were incubated without IKKε (-), or with purified wild-type IKKε (WT) or mutant IKKε (K38A) in kinase buffer and resolved by SDS-PAGE, followed by immunoblotting analysis with the indicated antibodies.
    Anti Tbk1 3504, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "IKKε phosphorylates kindlin-2 to induce invadopodia formation and promote colorectal cancer metastasis"

    Article Title: IKKε phosphorylates kindlin-2 to induce invadopodia formation and promote colorectal cancer metastasis

    Journal: Theranostics

    doi: 10.7150/thno.40397

    IKKε directly phosphorylates kindlin-2 at S159. (A) Slot blot analysis of the anti-phospho-kindlin-2-S159 antibody. The antibody was probed against an un-modified kindlin-2 peptide and phosphorylated peptides. (B) HCT116 cells were transfected with the indicated plasmids and whole cell extracts were immunoprecipitated (IP) with FLAG-conjugated M2 beads and then analyzed by immunoblotting (IB). (C)HCT116 cells were treated with the inhibitor IKK-3 at the indicated concentration for 24h and analyzed by immunoblotting. (D and E) HCT116 cells were transfected with the indicated siRNAs targeting IKKα, IKKβ, IKKε or TBK1. Cell lysates were analyzed by immunoblotting. (F) HCT116 cells were transfected with FLAG-tagged kindlin-2 and GFP-tagged IKKε for 48 h. Total cell lysates were immunoprecipitated using FLAG-conjugated M2 beads and analyzed by immunoblotting. (G) IKKε was immunoprecipitated using anti-IKKε antibody or the control antibody IgG from lysates of HCT116 cells. (H) HCT116 cells were transfected with the indicated plasmids and the whole cell extracts were prepared and analyzed by Co-IP assay flowed by immunoblotting with the indicated antibodies. (I) GST-tagged wild-type kindlin-2 or kindlin-2 (S159A) were incubated without IKKε (-), or with purified wild-type IKKε (WT) or mutant IKKε (K38A) in kinase buffer and resolved by SDS-PAGE, followed by immunoblotting analysis with the indicated antibodies.
    Figure Legend Snippet: IKKε directly phosphorylates kindlin-2 at S159. (A) Slot blot analysis of the anti-phospho-kindlin-2-S159 antibody. The antibody was probed against an un-modified kindlin-2 peptide and phosphorylated peptides. (B) HCT116 cells were transfected with the indicated plasmids and whole cell extracts were immunoprecipitated (IP) with FLAG-conjugated M2 beads and then analyzed by immunoblotting (IB). (C)HCT116 cells were treated with the inhibitor IKK-3 at the indicated concentration for 24h and analyzed by immunoblotting. (D and E) HCT116 cells were transfected with the indicated siRNAs targeting IKKα, IKKβ, IKKε or TBK1. Cell lysates were analyzed by immunoblotting. (F) HCT116 cells were transfected with FLAG-tagged kindlin-2 and GFP-tagged IKKε for 48 h. Total cell lysates were immunoprecipitated using FLAG-conjugated M2 beads and analyzed by immunoblotting. (G) IKKε was immunoprecipitated using anti-IKKε antibody or the control antibody IgG from lysates of HCT116 cells. (H) HCT116 cells were transfected with the indicated plasmids and the whole cell extracts were prepared and analyzed by Co-IP assay flowed by immunoblotting with the indicated antibodies. (I) GST-tagged wild-type kindlin-2 or kindlin-2 (S159A) were incubated without IKKε (-), or with purified wild-type IKKε (WT) or mutant IKKε (K38A) in kinase buffer and resolved by SDS-PAGE, followed by immunoblotting analysis with the indicated antibodies.

    Techniques Used: Dot Blot, Modification, Transfection, Immunoprecipitation, Western Blot, Concentration Assay, Co-Immunoprecipitation Assay, Incubation, Purification, Mutagenesis, SDS Page

    tbk1 nak d1b4 rabbit monoclonal  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc tbk1 nak d1b4 rabbit monoclonal
    Aberrant <t>TBK1</t> expression in CRC. (A) Differential TBK1 gene expression between CRC and normal samples from the TCGA database and three published microarray datasets (GSE117606, GSE68468, GSE37182). (B) WB analysis of TBK1 protein in tissue lysates from six randomly selected paired specimens. CRC tumors (T); normal tissues (N). (C) HE staining of normal tissue and CRC tissues. (D) IHC staining of TBK1 in representative normal and CRC tissues; scale bar = 50 µm. (E) Analysis of TBK1 IHC scores in normal tissues and CRC tissues and TBK1 IHC scores in CRC tissues with or without lymph node metastasis (LNM). * P< 0.05, *** P< 0.001, **** P< 0.0001.
    Tbk1 Nak D1b4 Rabbit Monoclonal, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression"

    Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.70742

    Aberrant TBK1 expression in CRC. (A) Differential TBK1 gene expression between CRC and normal samples from the TCGA database and three published microarray datasets (GSE117606, GSE68468, GSE37182). (B) WB analysis of TBK1 protein in tissue lysates from six randomly selected paired specimens. CRC tumors (T); normal tissues (N). (C) HE staining of normal tissue and CRC tissues. (D) IHC staining of TBK1 in representative normal and CRC tissues; scale bar = 50 µm. (E) Analysis of TBK1 IHC scores in normal tissues and CRC tissues and TBK1 IHC scores in CRC tissues with or without lymph node metastasis (LNM). * P< 0.05, *** P< 0.001, **** P< 0.0001.
    Figure Legend Snippet: Aberrant TBK1 expression in CRC. (A) Differential TBK1 gene expression between CRC and normal samples from the TCGA database and three published microarray datasets (GSE117606, GSE68468, GSE37182). (B) WB analysis of TBK1 protein in tissue lysates from six randomly selected paired specimens. CRC tumors (T); normal tissues (N). (C) HE staining of normal tissue and CRC tissues. (D) IHC staining of TBK1 in representative normal and CRC tissues; scale bar = 50 µm. (E) Analysis of TBK1 IHC scores in normal tissues and CRC tissues and TBK1 IHC scores in CRC tissues with or without lymph node metastasis (LNM). * P< 0.05, *** P< 0.001, **** P< 0.0001.

    Techniques Used: Expressing, Microarray, Staining, Immunohistochemistry

    Relationship between  TBK1  expression and clinicopathological factors in CRC patients (* P < 0.05)
    Figure Legend Snippet: Relationship between TBK1 expression and clinicopathological factors in CRC patients (* P < 0.05)

    Techniques Used: Expressing

    TBK1 restrained the mTORC1 signaling activation in CRC. (A) Correlation analysis of TBK1 and mTOR in normal tissues and CRC tissues based on the TCGA database. (B) GESA analyses of gene sets for mTORC1 signaling. NES: normalized enrichment score; FDR: false discovery rate. Negative NES indicates lower expression in TBK1-WT to TBK1-KO. (C) The expression of TBK1 and mTOR in CRC cells revealed by IF. Green: mTOR; red: TBK1; blue: DAPI; scale bar: 50 µm. (D) The expression of TBK1 in the five CRC cell lines (HCT116, HT-29, SW480, SW620, LOVO). TBK1 expression was quantified by the gray-scale value of straps. (E) HCT116 cells and SW480 were transfected with NC-siRNA, TBK1-si207 and TBK1-si1953, TBK1, T-mTOR, p-mTOR, AKT, p-AKT, T-S6K1, p-S6K1, 4E-BP1, p-4E-BP1 and GAPDH were analyzed by WB.
    Figure Legend Snippet: TBK1 restrained the mTORC1 signaling activation in CRC. (A) Correlation analysis of TBK1 and mTOR in normal tissues and CRC tissues based on the TCGA database. (B) GESA analyses of gene sets for mTORC1 signaling. NES: normalized enrichment score; FDR: false discovery rate. Negative NES indicates lower expression in TBK1-WT to TBK1-KO. (C) The expression of TBK1 and mTOR in CRC cells revealed by IF. Green: mTOR; red: TBK1; blue: DAPI; scale bar: 50 µm. (D) The expression of TBK1 in the five CRC cell lines (HCT116, HT-29, SW480, SW620, LOVO). TBK1 expression was quantified by the gray-scale value of straps. (E) HCT116 cells and SW480 were transfected with NC-siRNA, TBK1-si207 and TBK1-si1953, TBK1, T-mTOR, p-mTOR, AKT, p-AKT, T-S6K1, p-S6K1, 4E-BP1, p-4E-BP1 and GAPDH were analyzed by WB.

    Techniques Used: Activation Assay, Expressing, Transfection

    TBK1 depletion suppressed cell migration, proliferation and drug resistance in CRC. (A) Representative photographs of scratch wound assay of HCT116 and SW480 cells transfected with NC-siRNA or TBK1-siRNA. (scale bar = 50 µm). (B) The quantification analysis of the relative scratch area, mean ± SD (n=3). (C) Representative photographs of transwell assay of HCT116 and SW480 cells transfected with NC-siRNA or TBK1-siRNA. (scale bar = 50 µm). (D) The quantification of the migratory cell rate, mean ± SD (n=3). The relative cell viabilities of HCT116 (E) and SW480 (F) cells were tested with a CCK-8 assay, mean ± SD (n=3). CTL: transfected with NC-siRNA, 50 nM; DMSO: treated with DMSO; si-TBK1: transfected with TBK1-siRNA, 50 nM; 5-FU: treated with 5-FU; si-TBK1+5-FU: transfected with TBK1-siRNA+5-FU.
    Figure Legend Snippet: TBK1 depletion suppressed cell migration, proliferation and drug resistance in CRC. (A) Representative photographs of scratch wound assay of HCT116 and SW480 cells transfected with NC-siRNA or TBK1-siRNA. (scale bar = 50 µm). (B) The quantification analysis of the relative scratch area, mean ± SD (n=3). (C) Representative photographs of transwell assay of HCT116 and SW480 cells transfected with NC-siRNA or TBK1-siRNA. (scale bar = 50 µm). (D) The quantification of the migratory cell rate, mean ± SD (n=3). The relative cell viabilities of HCT116 (E) and SW480 (F) cells were tested with a CCK-8 assay, mean ± SD (n=3). CTL: transfected with NC-siRNA, 50 nM; DMSO: treated with DMSO; si-TBK1: transfected with TBK1-siRNA, 50 nM; 5-FU: treated with 5-FU; si-TBK1+5-FU: transfected with TBK1-siRNA+5-FU.

    Techniques Used: Migration, Scratch Wound Assay Assay, Transfection, Transwell Assay, CCK-8 Assay

    The inhibition of mTORC1 signaling increased the GLUT1 expression in CRC. (A) Correlation analysis of TBK1 and GLUT1 in normal colorectal and CRC tissues based on the TCGA database. (B) The lysates of HCT116 and SW480 transfected with two TBK1 siRNAs were blotted for GLUT1 and GAPDH. (C) HCT116 and SW480 cells transfected with NC-siRNA, TBK1-siRNA, vector plasmid and TBK1 WT plasmid as indicated, the cell lysis was immunoblotted. (D) The expression of TBK1, p-mTOR, p-S6K1, GLUT1and p-4E-BP1 were quantified. Data are mean ± SD (n=3) NC: negative control; TBK1-KD: TBK1 knockdown; TBK1-OE: TBK1 overexpression. (E) HCT116 and SW480 were treated with rapamycin (5 µM) for 12 h, the lysis was blotted and the GLUT1 expression was quantified. * P< 0.05, ** P< 0.01.
    Figure Legend Snippet: The inhibition of mTORC1 signaling increased the GLUT1 expression in CRC. (A) Correlation analysis of TBK1 and GLUT1 in normal colorectal and CRC tissues based on the TCGA database. (B) The lysates of HCT116 and SW480 transfected with two TBK1 siRNAs were blotted for GLUT1 and GAPDH. (C) HCT116 and SW480 cells transfected with NC-siRNA, TBK1-siRNA, vector plasmid and TBK1 WT plasmid as indicated, the cell lysis was immunoblotted. (D) The expression of TBK1, p-mTOR, p-S6K1, GLUT1and p-4E-BP1 were quantified. Data are mean ± SD (n=3) NC: negative control; TBK1-KD: TBK1 knockdown; TBK1-OE: TBK1 overexpression. (E) HCT116 and SW480 were treated with rapamycin (5 µM) for 12 h, the lysis was blotted and the GLUT1 expression was quantified. * P< 0.05, ** P< 0.01.

    Techniques Used: Inhibition, Expressing, Transfection, Plasmid Preparation, Lysis, Negative Control, Over Expression

    TBK1-induced autophagy inhibited GLUT1 degradation in CRC. (A) The mRNA levels of GLUT1, mTOR, Raptor and S6K1 in HCT116 cells transfected with NC-siRNA or TBK1-si207 for 24 h. (B) HCT116 cells were treated with cycloheximide (CHX) for 12 h after being transfected with NC-siRNA or TBK1-si207 for 24 h. GLUT1 and GAPDH of the cell lysates were blotted. The degradation curve is according to the relative GLIT1 grayscale value of each time point, and the bands were quantified and presented as the mean ± SD (n=3). (C) WB analysis of P62, GLUT1, LC3 II/I and GAPDH from whole-cell lysates. HCT116 cells were transfected with NC-siRNA, TBK1-si207, TBK1-si1953, vector plasmid and TBK1 WT plasmid as indicated. (D) WB analysis of TBK1, P62, GLUT1 and GAPDH in HCT116 cells with stable TBK1 knockdown or negative control. (E) Negative control (NC) and stable TBK1-knocked down HCT116 cells were separately transfected with NC-siRNA, ATG7-siRNA and TBC1D5-siRNA, and the whole cell lysates were immunoblotted for the indicated proteins.
    Figure Legend Snippet: TBK1-induced autophagy inhibited GLUT1 degradation in CRC. (A) The mRNA levels of GLUT1, mTOR, Raptor and S6K1 in HCT116 cells transfected with NC-siRNA or TBK1-si207 for 24 h. (B) HCT116 cells were treated with cycloheximide (CHX) for 12 h after being transfected with NC-siRNA or TBK1-si207 for 24 h. GLUT1 and GAPDH of the cell lysates were blotted. The degradation curve is according to the relative GLIT1 grayscale value of each time point, and the bands were quantified and presented as the mean ± SD (n=3). (C) WB analysis of P62, GLUT1, LC3 II/I and GAPDH from whole-cell lysates. HCT116 cells were transfected with NC-siRNA, TBK1-si207, TBK1-si1953, vector plasmid and TBK1 WT plasmid as indicated. (D) WB analysis of TBK1, P62, GLUT1 and GAPDH in HCT116 cells with stable TBK1 knockdown or negative control. (E) Negative control (NC) and stable TBK1-knocked down HCT116 cells were separately transfected with NC-siRNA, ATG7-siRNA and TBC1D5-siRNA, and the whole cell lysates were immunoblotted for the indicated proteins.

    Techniques Used: Transfection, Plasmid Preparation, Negative Control

    TBK1 facilitated the cell membrane localization of GLTU1 in CRC. (A) Representative IHC staining of TBK1 and GLUT1 in normal, dysplasia and CRC tissues (scale bar = 50 µm). Insets: Magnification of the boxed regions. (B) IF staining for GLUT1 in HCT116 cell with GFP-GLUT1 expression with indicated treatment. Scale bars: 8 µm. Insets: Magnification of the boxed regions. (C) Fluorescence images of HCT116 cells treated with 2-NBDG (100 µM) for 3h after NC-siRNA, TBK1-siRNA, Vector plasmid or TBK1 WT plasmid. The mean fluorescence intensities were quantified with Image J. Mean±SD, n=3, * P< 0.05.
    Figure Legend Snippet: TBK1 facilitated the cell membrane localization of GLTU1 in CRC. (A) Representative IHC staining of TBK1 and GLUT1 in normal, dysplasia and CRC tissues (scale bar = 50 µm). Insets: Magnification of the boxed regions. (B) IF staining for GLUT1 in HCT116 cell with GFP-GLUT1 expression with indicated treatment. Scale bars: 8 µm. Insets: Magnification of the boxed regions. (C) Fluorescence images of HCT116 cells treated with 2-NBDG (100 µM) for 3h after NC-siRNA, TBK1-siRNA, Vector plasmid or TBK1 WT plasmid. The mean fluorescence intensities were quantified with Image J. Mean±SD, n=3, * P< 0.05.

    Techniques Used: Immunohistochemistry, Staining, Expressing, Fluorescence, Plasmid Preparation

    TBK1 is a promising target for CRC treatment. (A) HCT116 and SW480 cells were treated with amlexanox (100 µM) or poly (I:C) (2 µg/ml) for 24 h, and the lysates were blotted for GLUT1. (B) The GLTU1 expression was quantified (mean±SD, n=3, * P< 0.05). (C) Representative image of tumors derived from NC-shRNA or TBK1-shRNA transfected HCT116 cells in nude mice (5/group). (D) Quantification of tumor volume and weight of NC and TBK1-KD groups (mean ± SD, n=5.) CCK-8 assay of HCT116 (E) and SW480 (F) cells treated as indicated. 5-FU, 5 µM; amlexanox (100 µM) for 24, 48 and 72 h. Data are mean ± SD (n=3). * P< 0.05, ** P< 0.01, *** P< 0.001.
    Figure Legend Snippet: TBK1 is a promising target for CRC treatment. (A) HCT116 and SW480 cells were treated with amlexanox (100 µM) or poly (I:C) (2 µg/ml) for 24 h, and the lysates were blotted for GLUT1. (B) The GLTU1 expression was quantified (mean±SD, n=3, * P< 0.05). (C) Representative image of tumors derived from NC-shRNA or TBK1-shRNA transfected HCT116 cells in nude mice (5/group). (D) Quantification of tumor volume and weight of NC and TBK1-KD groups (mean ± SD, n=5.) CCK-8 assay of HCT116 (E) and SW480 (F) cells treated as indicated. 5-FU, 5 µM; amlexanox (100 µM) for 24, 48 and 72 h. Data are mean ± SD (n=3). * P< 0.05, ** P< 0.01, *** P< 0.001.

    Techniques Used: Expressing, Derivative Assay, shRNA, Transfection, CCK-8 Assay

     TBK1  expression in colorectal tumor tissues and paracancer tissues
    Figure Legend Snippet: TBK1 expression in colorectal tumor tissues and paracancer tissues

    Techniques Used: Expressing

    tbk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc tbk1
    RocA increases the expressions of CCL5 and CXCL10 depending on <t>TBK1</t> and STING. A, DEGs related to the cGAS-STING signaling pathway were richened. A549, H1299, H1975, and LLC cells were treated with or without 25 nM of RocA in the presence or absence of 5 µM CYT387 (B) , 5 µM H-151 or 5 µM C-176 (C) , or 10 µM BAY11 for 24 h, and then the expressions of CCL5 and CXCL10 were analyzed by real-time PCR. Data were pooled from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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    1) Product Images from "Rocaglamide promotes the infiltration and antitumor immunity of NK cells by activating cGAS-STING signaling in non-small cell lung cancer"

    Article Title: Rocaglamide promotes the infiltration and antitumor immunity of NK cells by activating cGAS-STING signaling in non-small cell lung cancer

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.65019

    RocA increases the expressions of CCL5 and CXCL10 depending on TBK1 and STING. A, DEGs related to the cGAS-STING signaling pathway were richened. A549, H1299, H1975, and LLC cells were treated with or without 25 nM of RocA in the presence or absence of 5 µM CYT387 (B) , 5 µM H-151 or 5 µM C-176 (C) , or 10 µM BAY11 for 24 h, and then the expressions of CCL5 and CXCL10 were analyzed by real-time PCR. Data were pooled from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
    Figure Legend Snippet: RocA increases the expressions of CCL5 and CXCL10 depending on TBK1 and STING. A, DEGs related to the cGAS-STING signaling pathway were richened. A549, H1299, H1975, and LLC cells were treated with or without 25 nM of RocA in the presence or absence of 5 µM CYT387 (B) , 5 µM H-151 or 5 µM C-176 (C) , or 10 µM BAY11 for 24 h, and then the expressions of CCL5 and CXCL10 were analyzed by real-time PCR. Data were pooled from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

    Techniques Used: Real-time Polymerase Chain Reaction

    RocA activates the cGAS-STING signaling pathway and increases the expressions of CCL5 and CXCL10 depending on such pathway. A, A549, H1299, and H1975 cells were exposed to 25 nM of RocA for different durations (0, 1, 3, 6, 12, and 24 h), and then the expressions of cGAS, STING, pTBK1, TBK1, pIRF3, and IRF3 were detected by Western blotting analysis. B, A549, H1299, and H1975 cells were exposed to different concentrations (0, 12.5, 25, and 50 nM) of RocA for 24 h, and then the expressions of cGAS, pTBK1, TBK1, p65, and pp65 were detected by Western blotting analysis. C, A549, H1299, and H1975 cells were transfected with STING siRNA or negative control (NC) for 24 h and then exposed to 25 nM of RocA for 24 h, followed by the detection of STING, pTBK1, and TBK1 by Western blotting analysis. Data represented three independent experiments. E, A549, H1299, and H1975 cells were transfected with STING siRNA or NC for 24 h and then exposed to different concentrations (0, 12.5, and 25 nM) of RocA for 24 h, followed by the detection of CCL5 and CXCL10 by real-time PCR. F, A549, H1299, and H1975 cells were transfected with STING siRNA or NC for 24 h and then exposed to 25 nM of RocA for 24 h, followed by the analysis of NK cell migration. Data were pooled from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
    Figure Legend Snippet: RocA activates the cGAS-STING signaling pathway and increases the expressions of CCL5 and CXCL10 depending on such pathway. A, A549, H1299, and H1975 cells were exposed to 25 nM of RocA for different durations (0, 1, 3, 6, 12, and 24 h), and then the expressions of cGAS, STING, pTBK1, TBK1, pIRF3, and IRF3 were detected by Western blotting analysis. B, A549, H1299, and H1975 cells were exposed to different concentrations (0, 12.5, 25, and 50 nM) of RocA for 24 h, and then the expressions of cGAS, pTBK1, TBK1, p65, and pp65 were detected by Western blotting analysis. C, A549, H1299, and H1975 cells were transfected with STING siRNA or negative control (NC) for 24 h and then exposed to 25 nM of RocA for 24 h, followed by the detection of STING, pTBK1, and TBK1 by Western blotting analysis. Data represented three independent experiments. E, A549, H1299, and H1975 cells were transfected with STING siRNA or NC for 24 h and then exposed to different concentrations (0, 12.5, and 25 nM) of RocA for 24 h, followed by the detection of CCL5 and CXCL10 by real-time PCR. F, A549, H1299, and H1975 cells were transfected with STING siRNA or NC for 24 h and then exposed to 25 nM of RocA for 24 h, followed by the analysis of NK cell migration. Data were pooled from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

    Techniques Used: Western Blot, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Migration

    NK cell infiltration and tumor regression by RocA depend on STING. A, The expressions of STING, pTBK1, and TBK1 in STING WT and STING KO LLC cells were detected by Western blotting analysis. Data represented three independent experiments. STING WT and STING KO LLC cells were exposed to different concentrations (0, 12.5, and 25 nM) of RocA for 24 h, and then the expressions of CCL5 (B) and CXCL10 (C) were analyzed by real-time PCR. Data were pooled from three independent experiments. STING WT and STING KO LLC cells were subcutaneously inoculated onto the upper back of C57BL6 mice on day 0, and 1 mg/kg of RocA was administered by i.p. injection every 2 days from day 3. Tumor size was measured every 2 days (D, E) . Mice were sacrificed on day 18, and tumors were excised, photographed (F) , weighed (G) , and used to detect the proportions of NK cells (H-I) . Data represented three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, non-statistical significance.
    Figure Legend Snippet: NK cell infiltration and tumor regression by RocA depend on STING. A, The expressions of STING, pTBK1, and TBK1 in STING WT and STING KO LLC cells were detected by Western blotting analysis. Data represented three independent experiments. STING WT and STING KO LLC cells were exposed to different concentrations (0, 12.5, and 25 nM) of RocA for 24 h, and then the expressions of CCL5 (B) and CXCL10 (C) were analyzed by real-time PCR. Data were pooled from three independent experiments. STING WT and STING KO LLC cells were subcutaneously inoculated onto the upper back of C57BL6 mice on day 0, and 1 mg/kg of RocA was administered by i.p. injection every 2 days from day 3. Tumor size was measured every 2 days (D, E) . Mice were sacrificed on day 18, and tumors were excised, photographed (F) , weighed (G) , and used to detect the proportions of NK cells (H-I) . Data represented three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, non-statistical significance.

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Injection

    anti tbk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti tbk1
    (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 <t>TBK1,</t> 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.
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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

    phospho tbk1 nak  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho tbk1 nak
    Inhibition of STING impaired immunostimulatory effects of DM plasma derived small extracellular vesicles in PBMCs. (A) sEVs-stimulated PBMCs secreted less IFNβ release when STING antagonist H-151 (1μM) was present ((21.58 ± 5.45 vs. 28.34 ± 4.25) pg/mL; n = 6). (B) sEVs-stimulated PBMCs secreted less TNFα release when STING antagonist H-151 was present (434.8 ± 231.5 vs. 919.1 ± 325.7) pg/mL; n = 6). (C) sEVs-stimulated PBMCs secreted less IL6 release when STING antagonist H-151 was present ((611.5 ± 132.8 vs. 844.2 ± 180.3) pg/mL; n = 6). (D) STING antagonist H-151 suppressed DM plasma-derived sEV-induced STING phosphorylation and its downstream signaling pathway <t>TBK1,</t> IRF3, and NFκB phosphorylation in PBMCs. (E) Relative intensity of phosphorylated STING, phosphorylated TBK1, phosphorylated IRF3, and phosphorylated NFκB in PBMCs stimulated with/without DM derived sEVs in the presence/absence of STING antagonist H-151 (n = 3). Data in (A,B,C,E) represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups as indicated. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test. Comparison between two groups was analyzed by the Student t test.
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    1) Product Images from "Plasma-derived DNA containing-extracellular vesicles induce STING-mediated proinflammatory responses in dermatomyositis"

    Article Title: Plasma-derived DNA containing-extracellular vesicles induce STING-mediated proinflammatory responses in dermatomyositis

    Journal: Theranostics

    doi: 10.7150/thno.59152

    Inhibition of STING impaired immunostimulatory effects of DM plasma derived small extracellular vesicles in PBMCs. (A) sEVs-stimulated PBMCs secreted less IFNβ release when STING antagonist H-151 (1μM) was present ((21.58 ± 5.45 vs. 28.34 ± 4.25) pg/mL; n = 6). (B) sEVs-stimulated PBMCs secreted less TNFα release when STING antagonist H-151 was present (434.8 ± 231.5 vs. 919.1 ± 325.7) pg/mL; n = 6). (C) sEVs-stimulated PBMCs secreted less IL6 release when STING antagonist H-151 was present ((611.5 ± 132.8 vs. 844.2 ± 180.3) pg/mL; n = 6). (D) STING antagonist H-151 suppressed DM plasma-derived sEV-induced STING phosphorylation and its downstream signaling pathway TBK1, IRF3, and NFκB phosphorylation in PBMCs. (E) Relative intensity of phosphorylated STING, phosphorylated TBK1, phosphorylated IRF3, and phosphorylated NFκB in PBMCs stimulated with/without DM derived sEVs in the presence/absence of STING antagonist H-151 (n = 3). Data in (A,B,C,E) represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups as indicated. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test. Comparison between two groups was analyzed by the Student t test.
    Figure Legend Snippet: Inhibition of STING impaired immunostimulatory effects of DM plasma derived small extracellular vesicles in PBMCs. (A) sEVs-stimulated PBMCs secreted less IFNβ release when STING antagonist H-151 (1μM) was present ((21.58 ± 5.45 vs. 28.34 ± 4.25) pg/mL; n = 6). (B) sEVs-stimulated PBMCs secreted less TNFα release when STING antagonist H-151 was present (434.8 ± 231.5 vs. 919.1 ± 325.7) pg/mL; n = 6). (C) sEVs-stimulated PBMCs secreted less IL6 release when STING antagonist H-151 was present ((611.5 ± 132.8 vs. 844.2 ± 180.3) pg/mL; n = 6). (D) STING antagonist H-151 suppressed DM plasma-derived sEV-induced STING phosphorylation and its downstream signaling pathway TBK1, IRF3, and NFκB phosphorylation in PBMCs. (E) Relative intensity of phosphorylated STING, phosphorylated TBK1, phosphorylated IRF3, and phosphorylated NFκB in PBMCs stimulated with/without DM derived sEVs in the presence/absence of STING antagonist H-151 (n = 3). Data in (A,B,C,E) represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups as indicated. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test. Comparison between two groups was analyzed by the Student t test.

    Techniques Used: Inhibition, Derivative Assay

    Inhibition of TBK1 decreased DM plasma derived small extracellular vesicles' immunostimulatory effects in PBMCs. (A) Representative immunofluorescent staining images showing that sEVs derived from DM plasma induced phosphorylation of TBK1 in PBMCs. (Scale bar 100 µm) (B) Bar graphic depicting the relative intensity of phosphorylated TBK1 immunofluorescent staining in PBMCs with/without DM plasma-derived sEVs stimulation (n = 5). (C) TBK1 inhibitors suppressed DM plasma-derived sEVs induced TBK1 and IRF3 phosphorylation in PBMCs. (D) DM plasma-derived sEVs induced IFNβ release in PBMCs (11.40 ± 4.669 pg/mL, n = 5) when compared with untreated PBMCs (2.000 ± 0.7674 pg/mL, n = 5). 2.5 µM of Amlexanox (TBK1 inhibitor) pretreatment impaired sEVs-triggered IFNβ release in PBMCs (3.933 ± 2.002 pg/mL, n = 5); 2.5 µM of MRT67307 (TBK1 inhibitor) pretreatment impaired DM sEVs-triggered IFNβ release in PBMCs (4.067 ± 1.511 pg/mL, n = 5). Data in B represent median. Data in D represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups as indicated. Comparison between two groups was analyzed by the Student t test. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test.
    Figure Legend Snippet: Inhibition of TBK1 decreased DM plasma derived small extracellular vesicles' immunostimulatory effects in PBMCs. (A) Representative immunofluorescent staining images showing that sEVs derived from DM plasma induced phosphorylation of TBK1 in PBMCs. (Scale bar 100 µm) (B) Bar graphic depicting the relative intensity of phosphorylated TBK1 immunofluorescent staining in PBMCs with/without DM plasma-derived sEVs stimulation (n = 5). (C) TBK1 inhibitors suppressed DM plasma-derived sEVs induced TBK1 and IRF3 phosphorylation in PBMCs. (D) DM plasma-derived sEVs induced IFNβ release in PBMCs (11.40 ± 4.669 pg/mL, n = 5) when compared with untreated PBMCs (2.000 ± 0.7674 pg/mL, n = 5). 2.5 µM of Amlexanox (TBK1 inhibitor) pretreatment impaired sEVs-triggered IFNβ release in PBMCs (3.933 ± 2.002 pg/mL, n = 5); 2.5 µM of MRT67307 (TBK1 inhibitor) pretreatment impaired DM sEVs-triggered IFNβ release in PBMCs (4.067 ± 1.511 pg/mL, n = 5). Data in B represent median. Data in D represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups as indicated. Comparison between two groups was analyzed by the Student t test. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test.

    Techniques Used: Inhibition, Derivative Assay, Staining

    Digestion of DM plasma-derived small extracellular vesicles-captured DNA impaired their triggered STING signaling pathway activation in PBMCs. (A) The effects of DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without DNase I on STING phosphorylation and its downstream signaling pathway TBK1, and IRF3 phosphorylation in PBMCs. (B) The effects of DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without dsDNase on STING phosphorylation and its downstream signaling pathway TBK1, and IRF3 phosphorylation in PBMCs. Relative intensity of phosphorylated STING (C), phosphorylated TBK1(D), and phosphorylated IRF3 (E) in PBMCs stimulated by DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without DNase I (n = 3). Relative intensity of phosphorylated STING (F), phosphorylated TBK1(G), and phosphorylated IRF3 (H) in PBMCs stimulated by DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without dsDNase (n = 3). Data were represent mean ± SD. * P < 0.05,** P < 0.01 between groups as indicated. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test.
    Figure Legend Snippet: Digestion of DM plasma-derived small extracellular vesicles-captured DNA impaired their triggered STING signaling pathway activation in PBMCs. (A) The effects of DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without DNase I on STING phosphorylation and its downstream signaling pathway TBK1, and IRF3 phosphorylation in PBMCs. (B) The effects of DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without dsDNase on STING phosphorylation and its downstream signaling pathway TBK1, and IRF3 phosphorylation in PBMCs. Relative intensity of phosphorylated STING (C), phosphorylated TBK1(D), and phosphorylated IRF3 (E) in PBMCs stimulated by DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without DNase I (n = 3). Relative intensity of phosphorylated STING (F), phosphorylated TBK1(G), and phosphorylated IRF3 (H) in PBMCs stimulated by DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without dsDNase (n = 3). Data were represent mean ± SD. * P < 0.05,** P < 0.01 between groups as indicated. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test.

    Techniques Used: Derivative Assay, Activation Assay

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    Transcriptional regulation of T H 1 and T H 9 cell differentiation following STING activation. (A) Heatmap representing Ifng , Tbx21 , Irf1 , Gata3 , Il9 , Irf4 , Irf8 , Batf , Rorc , and Foxp3 mRNA expression (Z-score) from naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 72 hours. (B–D) WT naive CD4 T cells stimulated with cGAMP or control for 4(B) or 6(C, D) hours. (B) STING, p-STING, <t>TBK1</t> and p-TBK1 protein levels. (C) Cyt and Nuc localization of IRF3, p65 NF-κB and their phosphorylated forms. (D) Heatmap representing Ifna , Ifnb1 , Il6 , Tnfa , Ifit1 , Ifit2 , Mx2 and Cxcl10 mRNA expression (Log2FC) between CD4 T cells stimulated with cGAMP and control. Mean of replicates from three independent experiments. P values (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) determined by two-way analysis of variance (A) or unpaired t test (D). (E, F) RNA sequencing analysis from WT naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 16 hours. Biological replicates from three independent experiments. (E) Gene set enrichment analysis comparing expression in cGAMP-stimulated cells to control cells. Enrichment plot and score, Nom. P value and FDR q value shown for the three gene sets. (F) Heatmaps illustrating the hierarchical clustering of expression levels (rld values) of ISGs. cGAMP, 2′3′-cyclic guanosine monophosphate–adenosine monophosphate; Cyt, cytosolic; FDR, false discovery rate; ISG, interferon-stimulated gene; Nom, nominal; Nuc, nuclear; STING, stimulator of interferon genes; WT, wild type.
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    IKKε directly phosphorylates kindlin-2 at S159. (A) Slot blot analysis of the anti-phospho-kindlin-2-S159 antibody. The antibody was probed against an un-modified kindlin-2 peptide and phosphorylated peptides. (B) HCT116 cells were transfected with the indicated plasmids and whole cell extracts were immunoprecipitated (IP) with FLAG-conjugated M2 beads and then analyzed by immunoblotting (IB). (C)HCT116 cells were treated with the inhibitor IKK-3 at the indicated concentration for 24h and analyzed by immunoblotting. (D and E) HCT116 cells were transfected with the indicated siRNAs targeting IKKα, IKKβ, IKKε or <t>TBK1.</t> Cell lysates were analyzed by immunoblotting. (F) HCT116 cells were transfected with FLAG-tagged kindlin-2 and GFP-tagged IKKε for 48 h. Total cell lysates were immunoprecipitated using FLAG-conjugated M2 beads and analyzed by immunoblotting. (G) IKKε was immunoprecipitated using anti-IKKε antibody or the control antibody IgG from lysates of HCT116 cells. (H) HCT116 cells were transfected with the indicated plasmids and the whole cell extracts were prepared and analyzed by Co-IP assay flowed by immunoblotting with the indicated antibodies. (I) GST-tagged wild-type kindlin-2 or kindlin-2 (S159A) were incubated without IKKε (-), or with purified wild-type IKKε (WT) or mutant IKKε (K38A) in kinase buffer and resolved by SDS-PAGE, followed by immunoblotting analysis with the indicated antibodies.
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    Inhibition of STING impaired immunostimulatory effects of DM plasma derived small extracellular vesicles in PBMCs. (A) sEVs-stimulated PBMCs secreted less IFNβ release when STING antagonist H-151 (1μM) was present ((21.58 ± 5.45 vs. 28.34 ± 4.25) pg/mL; n = 6). (B) sEVs-stimulated PBMCs secreted less TNFα release when STING antagonist H-151 was present (434.8 ± 231.5 vs. 919.1 ± 325.7) pg/mL; n = 6). (C) sEVs-stimulated PBMCs secreted less IL6 release when STING antagonist H-151 was present ((611.5 ± 132.8 vs. 844.2 ± 180.3) pg/mL; n = 6). (D) STING antagonist H-151 suppressed DM plasma-derived sEV-induced STING phosphorylation and its downstream signaling pathway <t>TBK1,</t> IRF3, and NFκB phosphorylation in PBMCs. (E) Relative intensity of phosphorylated STING, phosphorylated TBK1, phosphorylated IRF3, and phosphorylated NFκB in PBMCs stimulated with/without DM derived sEVs in the presence/absence of STING antagonist H-151 (n = 3). Data in (A,B,C,E) represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups as indicated. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test. Comparison between two groups was analyzed by the Student t test.
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    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: Cell lysates were resuspended in 4× sample buffer (200 mM Tris, pH 6.8; 8% SDS; 40% glycerol; 0.4% bromophenol blue and 20% 2-mercaptoethanol) and heated at 95 ℃ for 5 min. Antibodies used were OPA1 (67589), DRP1 (8570), Mitofusin-1 (14793), Mitofusin-2 (11925), H2AX (2595), γH2AX (2577), H3 (9715), H3K4me1 (5326), H3K4me3 (9751), IRF3 (11904), p-IRF3 (S396) (29047), TBK1 (3504), p-TBK1 (S172) (5483), p65 (8242) and p-p65 (S536) (3033) from Cell Signaling; H3K4me2 (39679), H3K9me1 (39887), H3K9me2 (39683), H3K9me3 (39765), H3K27me1 (61015), H3K27me2 (39919), H3K27me3 (39155), H3K36me1 (61351), H3K36me2 (39255), H3K36me3 (61101) from Active Motif; α tubulin (sc-23948) from Santa Cruz; Lamin A/C (GTX101127) from GeneTex. α tubulin was in 1:20000 dilution.

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

    Transcriptional regulation of T H 1 and T H 9 cell differentiation following STING activation. (A) Heatmap representing Ifng , Tbx21 , Irf1 , Gata3 , Il9 , Irf4 , Irf8 , Batf , Rorc , and Foxp3 mRNA expression (Z-score) from naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 72 hours. (B–D) WT naive CD4 T cells stimulated with cGAMP or control for 4(B) or 6(C, D) hours. (B) STING, p-STING, TBK1 and p-TBK1 protein levels. (C) Cyt and Nuc localization of IRF3, p65 NF-κB and their phosphorylated forms. (D) Heatmap representing Ifna , Ifnb1 , Il6 , Tnfa , Ifit1 , Ifit2 , Mx2 and Cxcl10 mRNA expression (Log2FC) between CD4 T cells stimulated with cGAMP and control. Mean of replicates from three independent experiments. P values (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) determined by two-way analysis of variance (A) or unpaired t test (D). (E, F) RNA sequencing analysis from WT naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 16 hours. Biological replicates from three independent experiments. (E) Gene set enrichment analysis comparing expression in cGAMP-stimulated cells to control cells. Enrichment plot and score, Nom. P value and FDR q value shown for the three gene sets. (F) Heatmaps illustrating the hierarchical clustering of expression levels (rld values) of ISGs. cGAMP, 2′3′-cyclic guanosine monophosphate–adenosine monophosphate; Cyt, cytosolic; FDR, false discovery rate; ISG, interferon-stimulated gene; Nom, nominal; Nuc, nuclear; STING, stimulator of interferon genes; WT, wild type.

    Journal: Journal for Immunotherapy of Cancer

    Article Title: CD4 T cell-intrinsic STING signaling controls the differentiation and effector functions of T H 1 and T H 9 cells

    doi: 10.1136/jitc-2021-003459

    Figure Lengend Snippet: Transcriptional regulation of T H 1 and T H 9 cell differentiation following STING activation. (A) Heatmap representing Ifng , Tbx21 , Irf1 , Gata3 , Il9 , Irf4 , Irf8 , Batf , Rorc , and Foxp3 mRNA expression (Z-score) from naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 72 hours. (B–D) WT naive CD4 T cells stimulated with cGAMP or control for 4(B) or 6(C, D) hours. (B) STING, p-STING, TBK1 and p-TBK1 protein levels. (C) Cyt and Nuc localization of IRF3, p65 NF-κB and their phosphorylated forms. (D) Heatmap representing Ifna , Ifnb1 , Il6 , Tnfa , Ifit1 , Ifit2 , Mx2 and Cxcl10 mRNA expression (Log2FC) between CD4 T cells stimulated with cGAMP and control. Mean of replicates from three independent experiments. P values (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) determined by two-way analysis of variance (A) or unpaired t test (D). (E, F) RNA sequencing analysis from WT naive CD4 T cells stimulated with cGAMP or control and polarized into T H 1 or T H 9 cells for 16 hours. Biological replicates from three independent experiments. (E) Gene set enrichment analysis comparing expression in cGAMP-stimulated cells to control cells. Enrichment plot and score, Nom. P value and FDR q value shown for the three gene sets. (F) Heatmaps illustrating the hierarchical clustering of expression levels (rld values) of ISGs. cGAMP, 2′3′-cyclic guanosine monophosphate–adenosine monophosphate; Cyt, cytosolic; FDR, false discovery rate; ISG, interferon-stimulated gene; Nom, nominal; Nuc, nuclear; STING, stimulator of interferon genes; WT, wild type.

    Article Snippet: The following antibodies were purchased from Cell Signaling Technology (CST) and diluted 1:1000 in TBS-T 5% BSA: Phospho-STING (Ser 366, 19781), TBK1/NAK (clone D1B4, 3504) Phopho-TBK1 (Ser 172, clone D52C2, 5483), Phospho-IRF-3 (Ser 396, clone 4D4G, 4947), IRF3 (clone D83B9, 4302), Phospho-NF kappa B P65 (Ser 536, clone 93H1, 3033), NF kappa B P65 (clone D14E12, 8242), Phospho-p70S6K (Thr389, clone 108D2, 9234), and Phospho-S6 (Ser235/236, clone D57.2.2E, 4858).

    Techniques: Cell Differentiation, Activation Assay, Expressing, RNA Sequencing Assay

    IKKε directly phosphorylates kindlin-2 at S159. (A) Slot blot analysis of the anti-phospho-kindlin-2-S159 antibody. The antibody was probed against an un-modified kindlin-2 peptide and phosphorylated peptides. (B) HCT116 cells were transfected with the indicated plasmids and whole cell extracts were immunoprecipitated (IP) with FLAG-conjugated M2 beads and then analyzed by immunoblotting (IB). (C)HCT116 cells were treated with the inhibitor IKK-3 at the indicated concentration for 24h and analyzed by immunoblotting. (D and E) HCT116 cells were transfected with the indicated siRNAs targeting IKKα, IKKβ, IKKε or TBK1. Cell lysates were analyzed by immunoblotting. (F) HCT116 cells were transfected with FLAG-tagged kindlin-2 and GFP-tagged IKKε for 48 h. Total cell lysates were immunoprecipitated using FLAG-conjugated M2 beads and analyzed by immunoblotting. (G) IKKε was immunoprecipitated using anti-IKKε antibody or the control antibody IgG from lysates of HCT116 cells. (H) HCT116 cells were transfected with the indicated plasmids and the whole cell extracts were prepared and analyzed by Co-IP assay flowed by immunoblotting with the indicated antibodies. (I) GST-tagged wild-type kindlin-2 or kindlin-2 (S159A) were incubated without IKKε (-), or with purified wild-type IKKε (WT) or mutant IKKε (K38A) in kinase buffer and resolved by SDS-PAGE, followed by immunoblotting analysis with the indicated antibodies.

    Journal: Theranostics

    Article Title: IKKε phosphorylates kindlin-2 to induce invadopodia formation and promote colorectal cancer metastasis

    doi: 10.7150/thno.40397

    Figure Lengend Snippet: IKKε directly phosphorylates kindlin-2 at S159. (A) Slot blot analysis of the anti-phospho-kindlin-2-S159 antibody. The antibody was probed against an un-modified kindlin-2 peptide and phosphorylated peptides. (B) HCT116 cells were transfected with the indicated plasmids and whole cell extracts were immunoprecipitated (IP) with FLAG-conjugated M2 beads and then analyzed by immunoblotting (IB). (C)HCT116 cells were treated with the inhibitor IKK-3 at the indicated concentration for 24h and analyzed by immunoblotting. (D and E) HCT116 cells were transfected with the indicated siRNAs targeting IKKα, IKKβ, IKKε or TBK1. Cell lysates were analyzed by immunoblotting. (F) HCT116 cells were transfected with FLAG-tagged kindlin-2 and GFP-tagged IKKε for 48 h. Total cell lysates were immunoprecipitated using FLAG-conjugated M2 beads and analyzed by immunoblotting. (G) IKKε was immunoprecipitated using anti-IKKε antibody or the control antibody IgG from lysates of HCT116 cells. (H) HCT116 cells were transfected with the indicated plasmids and the whole cell extracts were prepared and analyzed by Co-IP assay flowed by immunoblotting with the indicated antibodies. (I) GST-tagged wild-type kindlin-2 or kindlin-2 (S159A) were incubated without IKKε (-), or with purified wild-type IKKε (WT) or mutant IKKε (K38A) in kinase buffer and resolved by SDS-PAGE, followed by immunoblotting analysis with the indicated antibodies.

    Article Snippet: The following antibodies were used in this study: anti-kindlin-2-S159 (CTM-152, PTM Biolabs) ; anti-kindlin-2 (K3269) and anti-FLAG (F3165) (Sigma-Aldrich); anti-IKKα (2682), anti-IKKβ (2678), anti-IKKε (2905), anti-TBK1 (3504) (Cell Signaling Technology); anti-GAPDH (sc-3223), anti-FISH (sc-376211) and anti-cortactin (sc-55579) (Santa Cruz); anti-IKKε (ab7891) (Abcam); anti-SH3PXD2A (18976-1-AP) (Proteintech); anti-MIG2/Kindlin-2 (NBP1-47745) (NOVUS).

    Techniques: Dot Blot, Modification, Transfection, Immunoprecipitation, Western Blot, Concentration Assay, Co-Immunoprecipitation Assay, Incubation, Purification, Mutagenesis, SDS Page

    Aberrant TBK1 expression in CRC. (A) Differential TBK1 gene expression between CRC and normal samples from the TCGA database and three published microarray datasets (GSE117606, GSE68468, GSE37182). (B) WB analysis of TBK1 protein in tissue lysates from six randomly selected paired specimens. CRC tumors (T); normal tissues (N). (C) HE staining of normal tissue and CRC tissues. (D) IHC staining of TBK1 in representative normal and CRC tissues; scale bar = 50 µm. (E) Analysis of TBK1 IHC scores in normal tissues and CRC tissues and TBK1 IHC scores in CRC tissues with or without lymph node metastasis (LNM). * P< 0.05, *** P< 0.001, **** P< 0.0001.

    Journal: International Journal of Biological Sciences

    Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

    doi: 10.7150/ijbs.70742

    Figure Lengend Snippet: Aberrant TBK1 expression in CRC. (A) Differential TBK1 gene expression between CRC and normal samples from the TCGA database and three published microarray datasets (GSE117606, GSE68468, GSE37182). (B) WB analysis of TBK1 protein in tissue lysates from six randomly selected paired specimens. CRC tumors (T); normal tissues (N). (C) HE staining of normal tissue and CRC tissues. (D) IHC staining of TBK1 in representative normal and CRC tissues; scale bar = 50 µm. (E) Analysis of TBK1 IHC scores in normal tissues and CRC tissues and TBK1 IHC scores in CRC tissues with or without lymph node metastasis (LNM). * P< 0.05, *** P< 0.001, **** P< 0.0001.

    Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

    Techniques: Expressing, Microarray, Staining, Immunohistochemistry

    Relationship between  TBK1  expression and clinicopathological factors in CRC patients (* P < 0.05)

    Journal: International Journal of Biological Sciences

    Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

    doi: 10.7150/ijbs.70742

    Figure Lengend Snippet: Relationship between TBK1 expression and clinicopathological factors in CRC patients (* P < 0.05)

    Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

    Techniques: Expressing

    TBK1 restrained the mTORC1 signaling activation in CRC. (A) Correlation analysis of TBK1 and mTOR in normal tissues and CRC tissues based on the TCGA database. (B) GESA analyses of gene sets for mTORC1 signaling. NES: normalized enrichment score; FDR: false discovery rate. Negative NES indicates lower expression in TBK1-WT to TBK1-KO. (C) The expression of TBK1 and mTOR in CRC cells revealed by IF. Green: mTOR; red: TBK1; blue: DAPI; scale bar: 50 µm. (D) The expression of TBK1 in the five CRC cell lines (HCT116, HT-29, SW480, SW620, LOVO). TBK1 expression was quantified by the gray-scale value of straps. (E) HCT116 cells and SW480 were transfected with NC-siRNA, TBK1-si207 and TBK1-si1953, TBK1, T-mTOR, p-mTOR, AKT, p-AKT, T-S6K1, p-S6K1, 4E-BP1, p-4E-BP1 and GAPDH were analyzed by WB.

    Journal: International Journal of Biological Sciences

    Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

    doi: 10.7150/ijbs.70742

    Figure Lengend Snippet: TBK1 restrained the mTORC1 signaling activation in CRC. (A) Correlation analysis of TBK1 and mTOR in normal tissues and CRC tissues based on the TCGA database. (B) GESA analyses of gene sets for mTORC1 signaling. NES: normalized enrichment score; FDR: false discovery rate. Negative NES indicates lower expression in TBK1-WT to TBK1-KO. (C) The expression of TBK1 and mTOR in CRC cells revealed by IF. Green: mTOR; red: TBK1; blue: DAPI; scale bar: 50 µm. (D) The expression of TBK1 in the five CRC cell lines (HCT116, HT-29, SW480, SW620, LOVO). TBK1 expression was quantified by the gray-scale value of straps. (E) HCT116 cells and SW480 were transfected with NC-siRNA, TBK1-si207 and TBK1-si1953, TBK1, T-mTOR, p-mTOR, AKT, p-AKT, T-S6K1, p-S6K1, 4E-BP1, p-4E-BP1 and GAPDH were analyzed by WB.

    Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

    Techniques: Activation Assay, Expressing, Transfection

    TBK1 depletion suppressed cell migration, proliferation and drug resistance in CRC. (A) Representative photographs of scratch wound assay of HCT116 and SW480 cells transfected with NC-siRNA or TBK1-siRNA. (scale bar = 50 µm). (B) The quantification analysis of the relative scratch area, mean ± SD (n=3). (C) Representative photographs of transwell assay of HCT116 and SW480 cells transfected with NC-siRNA or TBK1-siRNA. (scale bar = 50 µm). (D) The quantification of the migratory cell rate, mean ± SD (n=3). The relative cell viabilities of HCT116 (E) and SW480 (F) cells were tested with a CCK-8 assay, mean ± SD (n=3). CTL: transfected with NC-siRNA, 50 nM; DMSO: treated with DMSO; si-TBK1: transfected with TBK1-siRNA, 50 nM; 5-FU: treated with 5-FU; si-TBK1+5-FU: transfected with TBK1-siRNA+5-FU.

    Journal: International Journal of Biological Sciences

    Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

    doi: 10.7150/ijbs.70742

    Figure Lengend Snippet: TBK1 depletion suppressed cell migration, proliferation and drug resistance in CRC. (A) Representative photographs of scratch wound assay of HCT116 and SW480 cells transfected with NC-siRNA or TBK1-siRNA. (scale bar = 50 µm). (B) The quantification analysis of the relative scratch area, mean ± SD (n=3). (C) Representative photographs of transwell assay of HCT116 and SW480 cells transfected with NC-siRNA or TBK1-siRNA. (scale bar = 50 µm). (D) The quantification of the migratory cell rate, mean ± SD (n=3). The relative cell viabilities of HCT116 (E) and SW480 (F) cells were tested with a CCK-8 assay, mean ± SD (n=3). CTL: transfected with NC-siRNA, 50 nM; DMSO: treated with DMSO; si-TBK1: transfected with TBK1-siRNA, 50 nM; 5-FU: treated with 5-FU; si-TBK1+5-FU: transfected with TBK1-siRNA+5-FU.

    Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

    Techniques: Migration, Scratch Wound Assay Assay, Transfection, Transwell Assay, CCK-8 Assay

    The inhibition of mTORC1 signaling increased the GLUT1 expression in CRC. (A) Correlation analysis of TBK1 and GLUT1 in normal colorectal and CRC tissues based on the TCGA database. (B) The lysates of HCT116 and SW480 transfected with two TBK1 siRNAs were blotted for GLUT1 and GAPDH. (C) HCT116 and SW480 cells transfected with NC-siRNA, TBK1-siRNA, vector plasmid and TBK1 WT plasmid as indicated, the cell lysis was immunoblotted. (D) The expression of TBK1, p-mTOR, p-S6K1, GLUT1and p-4E-BP1 were quantified. Data are mean ± SD (n=3) NC: negative control; TBK1-KD: TBK1 knockdown; TBK1-OE: TBK1 overexpression. (E) HCT116 and SW480 were treated with rapamycin (5 µM) for 12 h, the lysis was blotted and the GLUT1 expression was quantified. * P< 0.05, ** P< 0.01.

    Journal: International Journal of Biological Sciences

    Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

    doi: 10.7150/ijbs.70742

    Figure Lengend Snippet: The inhibition of mTORC1 signaling increased the GLUT1 expression in CRC. (A) Correlation analysis of TBK1 and GLUT1 in normal colorectal and CRC tissues based on the TCGA database. (B) The lysates of HCT116 and SW480 transfected with two TBK1 siRNAs were blotted for GLUT1 and GAPDH. (C) HCT116 and SW480 cells transfected with NC-siRNA, TBK1-siRNA, vector plasmid and TBK1 WT plasmid as indicated, the cell lysis was immunoblotted. (D) The expression of TBK1, p-mTOR, p-S6K1, GLUT1and p-4E-BP1 were quantified. Data are mean ± SD (n=3) NC: negative control; TBK1-KD: TBK1 knockdown; TBK1-OE: TBK1 overexpression. (E) HCT116 and SW480 were treated with rapamycin (5 µM) for 12 h, the lysis was blotted and the GLUT1 expression was quantified. * P< 0.05, ** P< 0.01.

    Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

    Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Lysis, Negative Control, Over Expression

    TBK1-induced autophagy inhibited GLUT1 degradation in CRC. (A) The mRNA levels of GLUT1, mTOR, Raptor and S6K1 in HCT116 cells transfected with NC-siRNA or TBK1-si207 for 24 h. (B) HCT116 cells were treated with cycloheximide (CHX) for 12 h after being transfected with NC-siRNA or TBK1-si207 for 24 h. GLUT1 and GAPDH of the cell lysates were blotted. The degradation curve is according to the relative GLIT1 grayscale value of each time point, and the bands were quantified and presented as the mean ± SD (n=3). (C) WB analysis of P62, GLUT1, LC3 II/I and GAPDH from whole-cell lysates. HCT116 cells were transfected with NC-siRNA, TBK1-si207, TBK1-si1953, vector plasmid and TBK1 WT plasmid as indicated. (D) WB analysis of TBK1, P62, GLUT1 and GAPDH in HCT116 cells with stable TBK1 knockdown or negative control. (E) Negative control (NC) and stable TBK1-knocked down HCT116 cells were separately transfected with NC-siRNA, ATG7-siRNA and TBC1D5-siRNA, and the whole cell lysates were immunoblotted for the indicated proteins.

    Journal: International Journal of Biological Sciences

    Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

    doi: 10.7150/ijbs.70742

    Figure Lengend Snippet: TBK1-induced autophagy inhibited GLUT1 degradation in CRC. (A) The mRNA levels of GLUT1, mTOR, Raptor and S6K1 in HCT116 cells transfected with NC-siRNA or TBK1-si207 for 24 h. (B) HCT116 cells were treated with cycloheximide (CHX) for 12 h after being transfected with NC-siRNA or TBK1-si207 for 24 h. GLUT1 and GAPDH of the cell lysates were blotted. The degradation curve is according to the relative GLIT1 grayscale value of each time point, and the bands were quantified and presented as the mean ± SD (n=3). (C) WB analysis of P62, GLUT1, LC3 II/I and GAPDH from whole-cell lysates. HCT116 cells were transfected with NC-siRNA, TBK1-si207, TBK1-si1953, vector plasmid and TBK1 WT plasmid as indicated. (D) WB analysis of TBK1, P62, GLUT1 and GAPDH in HCT116 cells with stable TBK1 knockdown or negative control. (E) Negative control (NC) and stable TBK1-knocked down HCT116 cells were separately transfected with NC-siRNA, ATG7-siRNA and TBC1D5-siRNA, and the whole cell lysates were immunoblotted for the indicated proteins.

    Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

    Techniques: Transfection, Plasmid Preparation, Negative Control

    TBK1 facilitated the cell membrane localization of GLTU1 in CRC. (A) Representative IHC staining of TBK1 and GLUT1 in normal, dysplasia and CRC tissues (scale bar = 50 µm). Insets: Magnification of the boxed regions. (B) IF staining for GLUT1 in HCT116 cell with GFP-GLUT1 expression with indicated treatment. Scale bars: 8 µm. Insets: Magnification of the boxed regions. (C) Fluorescence images of HCT116 cells treated with 2-NBDG (100 µM) for 3h after NC-siRNA, TBK1-siRNA, Vector plasmid or TBK1 WT plasmid. The mean fluorescence intensities were quantified with Image J. Mean±SD, n=3, * P< 0.05.

    Journal: International Journal of Biological Sciences

    Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

    doi: 10.7150/ijbs.70742

    Figure Lengend Snippet: TBK1 facilitated the cell membrane localization of GLTU1 in CRC. (A) Representative IHC staining of TBK1 and GLUT1 in normal, dysplasia and CRC tissues (scale bar = 50 µm). Insets: Magnification of the boxed regions. (B) IF staining for GLUT1 in HCT116 cell with GFP-GLUT1 expression with indicated treatment. Scale bars: 8 µm. Insets: Magnification of the boxed regions. (C) Fluorescence images of HCT116 cells treated with 2-NBDG (100 µM) for 3h after NC-siRNA, TBK1-siRNA, Vector plasmid or TBK1 WT plasmid. The mean fluorescence intensities were quantified with Image J. Mean±SD, n=3, * P< 0.05.

    Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

    Techniques: Immunohistochemistry, Staining, Expressing, Fluorescence, Plasmid Preparation

    TBK1 is a promising target for CRC treatment. (A) HCT116 and SW480 cells were treated with amlexanox (100 µM) or poly (I:C) (2 µg/ml) for 24 h, and the lysates were blotted for GLUT1. (B) The GLTU1 expression was quantified (mean±SD, n=3, * P< 0.05). (C) Representative image of tumors derived from NC-shRNA or TBK1-shRNA transfected HCT116 cells in nude mice (5/group). (D) Quantification of tumor volume and weight of NC and TBK1-KD groups (mean ± SD, n=5.) CCK-8 assay of HCT116 (E) and SW480 (F) cells treated as indicated. 5-FU, 5 µM; amlexanox (100 µM) for 24, 48 and 72 h. Data are mean ± SD (n=3). * P< 0.05, ** P< 0.01, *** P< 0.001.

    Journal: International Journal of Biological Sciences

    Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

    doi: 10.7150/ijbs.70742

    Figure Lengend Snippet: TBK1 is a promising target for CRC treatment. (A) HCT116 and SW480 cells were treated with amlexanox (100 µM) or poly (I:C) (2 µg/ml) for 24 h, and the lysates were blotted for GLUT1. (B) The GLTU1 expression was quantified (mean±SD, n=3, * P< 0.05). (C) Representative image of tumors derived from NC-shRNA or TBK1-shRNA transfected HCT116 cells in nude mice (5/group). (D) Quantification of tumor volume and weight of NC and TBK1-KD groups (mean ± SD, n=5.) CCK-8 assay of HCT116 (E) and SW480 (F) cells treated as indicated. 5-FU, 5 µM; amlexanox (100 µM) for 24, 48 and 72 h. Data are mean ± SD (n=3). * P< 0.05, ** P< 0.01, *** P< 0.001.

    Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

    Techniques: Expressing, Derivative Assay, shRNA, Transfection, CCK-8 Assay

     TBK1  expression in colorectal tumor tissues and paracancer tissues

    Journal: International Journal of Biological Sciences

    Article Title: TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

    doi: 10.7150/ijbs.70742

    Figure Lengend Snippet: TBK1 expression in colorectal tumor tissues and paracancer tissues

    Article Snippet: TBK1/NAK (D1B4) rabbit monoclonal (#3504), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), 4E-BP1 (#9452), phosoho-4E-BP1(Thr37/46) (#2855), LC3A/B (#12741) and phospho-p70 S6 Kinase (Thr389) (D5U1O) rabbit monoclonal antibodies (#97596) for immunoblotting were obtained from Cell Signaling Technology (CSTS, Danvers, MA).

    Techniques: Expressing

    Inhibition of STING impaired immunostimulatory effects of DM plasma derived small extracellular vesicles in PBMCs. (A) sEVs-stimulated PBMCs secreted less IFNβ release when STING antagonist H-151 (1μM) was present ((21.58 ± 5.45 vs. 28.34 ± 4.25) pg/mL; n = 6). (B) sEVs-stimulated PBMCs secreted less TNFα release when STING antagonist H-151 was present (434.8 ± 231.5 vs. 919.1 ± 325.7) pg/mL; n = 6). (C) sEVs-stimulated PBMCs secreted less IL6 release when STING antagonist H-151 was present ((611.5 ± 132.8 vs. 844.2 ± 180.3) pg/mL; n = 6). (D) STING antagonist H-151 suppressed DM plasma-derived sEV-induced STING phosphorylation and its downstream signaling pathway TBK1, IRF3, and NFκB phosphorylation in PBMCs. (E) Relative intensity of phosphorylated STING, phosphorylated TBK1, phosphorylated IRF3, and phosphorylated NFκB in PBMCs stimulated with/without DM derived sEVs in the presence/absence of STING antagonist H-151 (n = 3). Data in (A,B,C,E) represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups as indicated. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test. Comparison between two groups was analyzed by the Student t test.

    Journal: Theranostics

    Article Title: Plasma-derived DNA containing-extracellular vesicles induce STING-mediated proinflammatory responses in dermatomyositis

    doi: 10.7150/thno.59152

    Figure Lengend Snippet: Inhibition of STING impaired immunostimulatory effects of DM plasma derived small extracellular vesicles in PBMCs. (A) sEVs-stimulated PBMCs secreted less IFNβ release when STING antagonist H-151 (1μM) was present ((21.58 ± 5.45 vs. 28.34 ± 4.25) pg/mL; n = 6). (B) sEVs-stimulated PBMCs secreted less TNFα release when STING antagonist H-151 was present (434.8 ± 231.5 vs. 919.1 ± 325.7) pg/mL; n = 6). (C) sEVs-stimulated PBMCs secreted less IL6 release when STING antagonist H-151 was present ((611.5 ± 132.8 vs. 844.2 ± 180.3) pg/mL; n = 6). (D) STING antagonist H-151 suppressed DM plasma-derived sEV-induced STING phosphorylation and its downstream signaling pathway TBK1, IRF3, and NFκB phosphorylation in PBMCs. (E) Relative intensity of phosphorylated STING, phosphorylated TBK1, phosphorylated IRF3, and phosphorylated NFκB in PBMCs stimulated with/without DM derived sEVs in the presence/absence of STING antagonist H-151 (n = 3). Data in (A,B,C,E) represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups as indicated. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test. Comparison between two groups was analyzed by the Student t test.

    Article Snippet: Phospho-STING (Ser366) (D7C3S) rabbit antibody, Phospho-STING (Ser366) (D8K6H) rabbit antibody, Phospho-STING (Ser365) (D8F4W) rabbit antibody, STING (D2P2F) rabbit antibody, phospho-TBK1/NAK (Ser 172) (D52C2) rabbit antibody, TBK1/NAK (D1B4) rabbit antibody, phospho-IRF-3 (Ser396) (D601M) rabbit antibody, IRF-3 (D83B9) rabbit antibody, and β-Actin (8H10D10) mouse antibody were obtained from Cell Signaling Technology Company (Danvers, MA).

    Techniques: Inhibition, Derivative Assay

    Inhibition of TBK1 decreased DM plasma derived small extracellular vesicles' immunostimulatory effects in PBMCs. (A) Representative immunofluorescent staining images showing that sEVs derived from DM plasma induced phosphorylation of TBK1 in PBMCs. (Scale bar 100 µm) (B) Bar graphic depicting the relative intensity of phosphorylated TBK1 immunofluorescent staining in PBMCs with/without DM plasma-derived sEVs stimulation (n = 5). (C) TBK1 inhibitors suppressed DM plasma-derived sEVs induced TBK1 and IRF3 phosphorylation in PBMCs. (D) DM plasma-derived sEVs induced IFNβ release in PBMCs (11.40 ± 4.669 pg/mL, n = 5) when compared with untreated PBMCs (2.000 ± 0.7674 pg/mL, n = 5). 2.5 µM of Amlexanox (TBK1 inhibitor) pretreatment impaired sEVs-triggered IFNβ release in PBMCs (3.933 ± 2.002 pg/mL, n = 5); 2.5 µM of MRT67307 (TBK1 inhibitor) pretreatment impaired DM sEVs-triggered IFNβ release in PBMCs (4.067 ± 1.511 pg/mL, n = 5). Data in B represent median. Data in D represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups as indicated. Comparison between two groups was analyzed by the Student t test. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test.

    Journal: Theranostics

    Article Title: Plasma-derived DNA containing-extracellular vesicles induce STING-mediated proinflammatory responses in dermatomyositis

    doi: 10.7150/thno.59152

    Figure Lengend Snippet: Inhibition of TBK1 decreased DM plasma derived small extracellular vesicles' immunostimulatory effects in PBMCs. (A) Representative immunofluorescent staining images showing that sEVs derived from DM plasma induced phosphorylation of TBK1 in PBMCs. (Scale bar 100 µm) (B) Bar graphic depicting the relative intensity of phosphorylated TBK1 immunofluorescent staining in PBMCs with/without DM plasma-derived sEVs stimulation (n = 5). (C) TBK1 inhibitors suppressed DM plasma-derived sEVs induced TBK1 and IRF3 phosphorylation in PBMCs. (D) DM plasma-derived sEVs induced IFNβ release in PBMCs (11.40 ± 4.669 pg/mL, n = 5) when compared with untreated PBMCs (2.000 ± 0.7674 pg/mL, n = 5). 2.5 µM of Amlexanox (TBK1 inhibitor) pretreatment impaired sEVs-triggered IFNβ release in PBMCs (3.933 ± 2.002 pg/mL, n = 5); 2.5 µM of MRT67307 (TBK1 inhibitor) pretreatment impaired DM sEVs-triggered IFNβ release in PBMCs (4.067 ± 1.511 pg/mL, n = 5). Data in B represent median. Data in D represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups as indicated. Comparison between two groups was analyzed by the Student t test. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test.

    Article Snippet: Phospho-STING (Ser366) (D7C3S) rabbit antibody, Phospho-STING (Ser366) (D8K6H) rabbit antibody, Phospho-STING (Ser365) (D8F4W) rabbit antibody, STING (D2P2F) rabbit antibody, phospho-TBK1/NAK (Ser 172) (D52C2) rabbit antibody, TBK1/NAK (D1B4) rabbit antibody, phospho-IRF-3 (Ser396) (D601M) rabbit antibody, IRF-3 (D83B9) rabbit antibody, and β-Actin (8H10D10) mouse antibody were obtained from Cell Signaling Technology Company (Danvers, MA).

    Techniques: Inhibition, Derivative Assay, Staining

    Digestion of DM plasma-derived small extracellular vesicles-captured DNA impaired their triggered STING signaling pathway activation in PBMCs. (A) The effects of DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without DNase I on STING phosphorylation and its downstream signaling pathway TBK1, and IRF3 phosphorylation in PBMCs. (B) The effects of DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without dsDNase on STING phosphorylation and its downstream signaling pathway TBK1, and IRF3 phosphorylation in PBMCs. Relative intensity of phosphorylated STING (C), phosphorylated TBK1(D), and phosphorylated IRF3 (E) in PBMCs stimulated by DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without DNase I (n = 3). Relative intensity of phosphorylated STING (F), phosphorylated TBK1(G), and phosphorylated IRF3 (H) in PBMCs stimulated by DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without dsDNase (n = 3). Data were represent mean ± SD. * P < 0.05,** P < 0.01 between groups as indicated. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test.

    Journal: Theranostics

    Article Title: Plasma-derived DNA containing-extracellular vesicles induce STING-mediated proinflammatory responses in dermatomyositis

    doi: 10.7150/thno.59152

    Figure Lengend Snippet: Digestion of DM plasma-derived small extracellular vesicles-captured DNA impaired their triggered STING signaling pathway activation in PBMCs. (A) The effects of DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without DNase I on STING phosphorylation and its downstream signaling pathway TBK1, and IRF3 phosphorylation in PBMCs. (B) The effects of DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without dsDNase on STING phosphorylation and its downstream signaling pathway TBK1, and IRF3 phosphorylation in PBMCs. Relative intensity of phosphorylated STING (C), phosphorylated TBK1(D), and phosphorylated IRF3 (E) in PBMCs stimulated by DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without DNase I (n = 3). Relative intensity of phosphorylated STING (F), phosphorylated TBK1(G), and phosphorylated IRF3 (H) in PBMCs stimulated by DM plasma derived sEVs pretreated in the presence/absence of 0.075% Triton X-100 with/without dsDNase (n = 3). Data were represent mean ± SD. * P < 0.05,** P < 0.01 between groups as indicated. Comparison among three or more groups was performed using ANOVA, followed by Student-Newman-Keuls test.

    Article Snippet: Phospho-STING (Ser366) (D7C3S) rabbit antibody, Phospho-STING (Ser366) (D8K6H) rabbit antibody, Phospho-STING (Ser365) (D8F4W) rabbit antibody, STING (D2P2F) rabbit antibody, phospho-TBK1/NAK (Ser 172) (D52C2) rabbit antibody, TBK1/NAK (D1B4) rabbit antibody, phospho-IRF-3 (Ser396) (D601M) rabbit antibody, IRF-3 (D83B9) rabbit antibody, and β-Actin (8H10D10) mouse antibody were obtained from Cell Signaling Technology Company (Danvers, MA).

    Techniques: Derivative Assay, Activation Assay