tlr9 agonist cpg dna  (Hycult Biotech)


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    Hycult Biotech tlr9 agonist cpg dna
    Tlr9 Agonist Cpg Dna, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tlr9 agonist cpg dna  (Hycult Biotech)


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    Hycult Biotech tlr9 agonist cpg dna
    Tlr9 Agonist Cpg Dna, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tlr9 agonist cpg dna  (Hycult Biotech)


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    Hycult Biotech tlr9 agonist cpg dna
    Identification and distribution of <t>TLR9-expressing</t> cells in BM specimens from patients with OLP. ( A ) Representative images of immunofluorescence staining of TLR9 (red) in BM specimens from patients with OLP and HK. Scale bars, 100 μm. ( B ) Number of TLR9-positive cells in BM specimens from patients with OLP (n = 20) and HK (n = 12). ( C ) Representative images of double immunofluorescence staining for TLR9 (red), CD20 (blue), and CD123 (green) in BM specimens from patients with OLP. Scale bars, 100 μm. ( D ) Number of TLR9-expressing CD20- and CD123-positive cells in BM specimens from patients with OLP (n = 20). In ( B ) and ( D ), data are shown as box plots. Each box represents the upper and lower interquartile range. Lines inside the boxes represent the median. Symbols represent individual subjects. *** P < 0.001, **** P < 0.0001, by one-way ANOVA ( B ) or Mann–Whitney U tests ( D ). See Fig. for other definitions.
    Tlr9 Agonist Cpg Dna, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Toll-like receptor 9-positive plasmacytoid dendritic cells promote Th17 immune responses in oral lichen planus stimulated by epithelium-derived cathepsin K"

    Article Title: Toll-like receptor 9-positive plasmacytoid dendritic cells promote Th17 immune responses in oral lichen planus stimulated by epithelium-derived cathepsin K

    Journal: Scientific Reports

    doi: 10.1038/s41598-023-46090-3

    Identification and distribution of TLR9-expressing cells in BM specimens from patients with OLP. ( A ) Representative images of immunofluorescence staining of TLR9 (red) in BM specimens from patients with OLP and HK. Scale bars, 100 μm. ( B ) Number of TLR9-positive cells in BM specimens from patients with OLP (n = 20) and HK (n = 12). ( C ) Representative images of double immunofluorescence staining for TLR9 (red), CD20 (blue), and CD123 (green) in BM specimens from patients with OLP. Scale bars, 100 μm. ( D ) Number of TLR9-expressing CD20- and CD123-positive cells in BM specimens from patients with OLP (n = 20). In ( B ) and ( D ), data are shown as box plots. Each box represents the upper and lower interquartile range. Lines inside the boxes represent the median. Symbols represent individual subjects. *** P < 0.001, **** P < 0.0001, by one-way ANOVA ( B ) or Mann–Whitney U tests ( D ). See Fig. for other definitions.
    Figure Legend Snippet: Identification and distribution of TLR9-expressing cells in BM specimens from patients with OLP. ( A ) Representative images of immunofluorescence staining of TLR9 (red) in BM specimens from patients with OLP and HK. Scale bars, 100 μm. ( B ) Number of TLR9-positive cells in BM specimens from patients with OLP (n = 20) and HK (n = 12). ( C ) Representative images of double immunofluorescence staining for TLR9 (red), CD20 (blue), and CD123 (green) in BM specimens from patients with OLP. Scale bars, 100 μm. ( D ) Number of TLR9-expressing CD20- and CD123-positive cells in BM specimens from patients with OLP (n = 20). In ( B ) and ( D ), data are shown as box plots. Each box represents the upper and lower interquartile range. Lines inside the boxes represent the median. Symbols represent individual subjects. *** P < 0.001, **** P < 0.0001, by one-way ANOVA ( B ) or Mann–Whitney U tests ( D ). See Fig. for other definitions.

    Techniques Used: Expressing, Immunofluorescence, Staining, Double Immunofluorescence Staining, MANN-WHITNEY

    Association of CD123 + plasmacytoid dendritic cells (pDCs) with Th17 cell differentiation. ( A ) Schematic illustration of the experimental procedures; human CD123 + pDCs were extracted from peripheral blood mononuclear cells (PBMCs) and then stimulated by the Toll-like receptor 9 (TLR9) agonist CpG DNA, TLR9 inhibitor iODN, and/or CTSK in vitro. ( B ) Production of Th17-related cytokines (IL-6, IL-23, and TGF-β) in CD123 + pDCs stimulated with CpG DNA, iODN, and/or CTSK (n = 3/sample). Bars show the mean ± SD. Symbols represent individual subjects. * P < 0.05, ** P < 0.01, by one-way ANOVA. ( C ) Representative images of immunofluorescence staining for CD4 (green), RORγt (pink) and DAPI (blue) in BM specimens from patients with OLP and HK. Scale bars, 100 μm. ( D ), ( E ) Quantification of CD4 + RORγt + Th17 cells in BM specimens from patients with OLP (n = 20) and HK (n = 12). ( F ) Correlation among the numbers of CTSK + cells, TLR9 + CD123 + pDCs, and CD4 + RORγt + Th17 cells in BM specimens from patients with OLP (n = 20). The correlation coefficients were determined by Spearman’s rank correlations. ( G ) Number of TLR9 + CD123 + pDCs and CD4 + RORγt + Th17 cells in BM specimens from patients with reticular type OLP (n = 9) and erosive type OLP (n = 11). In (D ), ( E ) and ( G ) data are shown as box plots. Each box represents the upper and lower interquartile range. Lines inside the boxes represent the median. Symbols represent individual subjects. ** P < 0.01, *** P < 0.001, **** P < 0.0001 by one-way ANOVA ( D and E ) or Mann–Whitney U tests ( G ). See Fig. for other definitions.
    Figure Legend Snippet: Association of CD123 + plasmacytoid dendritic cells (pDCs) with Th17 cell differentiation. ( A ) Schematic illustration of the experimental procedures; human CD123 + pDCs were extracted from peripheral blood mononuclear cells (PBMCs) and then stimulated by the Toll-like receptor 9 (TLR9) agonist CpG DNA, TLR9 inhibitor iODN, and/or CTSK in vitro. ( B ) Production of Th17-related cytokines (IL-6, IL-23, and TGF-β) in CD123 + pDCs stimulated with CpG DNA, iODN, and/or CTSK (n = 3/sample). Bars show the mean ± SD. Symbols represent individual subjects. * P < 0.05, ** P < 0.01, by one-way ANOVA. ( C ) Representative images of immunofluorescence staining for CD4 (green), RORγt (pink) and DAPI (blue) in BM specimens from patients with OLP and HK. Scale bars, 100 μm. ( D ), ( E ) Quantification of CD4 + RORγt + Th17 cells in BM specimens from patients with OLP (n = 20) and HK (n = 12). ( F ) Correlation among the numbers of CTSK + cells, TLR9 + CD123 + pDCs, and CD4 + RORγt + Th17 cells in BM specimens from patients with OLP (n = 20). The correlation coefficients were determined by Spearman’s rank correlations. ( G ) Number of TLR9 + CD123 + pDCs and CD4 + RORγt + Th17 cells in BM specimens from patients with reticular type OLP (n = 9) and erosive type OLP (n = 11). In (D ), ( E ) and ( G ) data are shown as box plots. Each box represents the upper and lower interquartile range. Lines inside the boxes represent the median. Symbols represent individual subjects. ** P < 0.01, *** P < 0.001, **** P < 0.0001 by one-way ANOVA ( D and E ) or Mann–Whitney U tests ( G ). See Fig. for other definitions.

    Techniques Used: Cell Differentiation, In Vitro, Immunofluorescence, Staining, MANN-WHITNEY

    Single cell RNA-sequencing (scRNA-seq) analysis in BM specimens from patients with OLP. ( A ) Uniform manifold approximation and projection (UMAP) of CD45 + immune cells colored by cell types. ( B ) UMAP colored by TLR9 expression. ( C ) The violin plots showing the expression pattern of the selected cell markers in each of the clusters. ( D ) The percentage of CD45 + immune cells in TLR9 + cells. ( E ) KEGG pathways of upregulated DEGs in TLR9 + pDC compared with TLR9 − pDC. ( F ) GO analysis of DEGs upregulated in TLR9 + pDC compared with TLR9 − pDC.
    Figure Legend Snippet: Single cell RNA-sequencing (scRNA-seq) analysis in BM specimens from patients with OLP. ( A ) Uniform manifold approximation and projection (UMAP) of CD45 + immune cells colored by cell types. ( B ) UMAP colored by TLR9 expression. ( C ) The violin plots showing the expression pattern of the selected cell markers in each of the clusters. ( D ) The percentage of CD45 + immune cells in TLR9 + cells. ( E ) KEGG pathways of upregulated DEGs in TLR9 + pDC compared with TLR9 − pDC. ( F ) GO analysis of DEGs upregulated in TLR9 + pDC compared with TLR9 − pDC.

    Techniques Used: RNA Sequencing Assay, Expressing

    Schematic model of CTSK-induced TLR9 signaling in pDCs, leading to inflammation in OLP. TLR9 expressed on CD123-positive pDCs in subepithelial layers recognizes viral DNA. CTSK released from the LE activates TLR9 signaling in pDCs and their production of Th17-related cytokines, including IL-6, IL-23, and TGFβ, which leads to Th17-associated inflammation in OLP. See Fig. for other definitions.
    Figure Legend Snippet: Schematic model of CTSK-induced TLR9 signaling in pDCs, leading to inflammation in OLP. TLR9 expressed on CD123-positive pDCs in subepithelial layers recognizes viral DNA. CTSK released from the LE activates TLR9 signaling in pDCs and their production of Th17-related cytokines, including IL-6, IL-23, and TGFβ, which leads to Th17-associated inflammation in OLP. See Fig. for other definitions.

    Techniques Used:

    tlr9 agonist cpg dna  (Hycult Biotech)


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    Hycult Biotech tlr9 agonist cpg dna
    Tlr9 Agonist Cpg Dna, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tlr9 agonist cpg a  (Hycult Biotech)


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    Hycult Biotech tlr9 agonist cpg a
    AMA effects on the production of type I IFNs and phosphorylation of IRF7 in <t>TLR9-activated</t> GEN2.2 cells. Cells were stimulated with the TLR9 agonist <t>CpG-A</t> (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A). The level of secreted IFN-α cytokine was determined by ELISA (B). Cells were stimulated for 30 min as described above then the levels of the phosphorylated (p-IRF7) and native form of IRF7 were determined by Western blotting (C, D). Bars represent the means ± SD of three individual experiments (A, B, D) and a representative blot is shown (C). ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05, ## p < 0.01, #### p < 0.0001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.
    Tlr9 Agonist Cpg A, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells"

    Article Title: Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells

    Journal: Redox Biology

    doi: 10.1016/j.redox.2017.07.016

    AMA effects on the production of type I IFNs and phosphorylation of IRF7 in TLR9-activated GEN2.2 cells. Cells were stimulated with the TLR9 agonist CpG-A (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A). The level of secreted IFN-α cytokine was determined by ELISA (B). Cells were stimulated for 30 min as described above then the levels of the phosphorylated (p-IRF7) and native form of IRF7 were determined by Western blotting (C, D). Bars represent the means ± SD of three individual experiments (A, B, D) and a representative blot is shown (C). ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05, ## p < 0.01, #### p < 0.0001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.
    Figure Legend Snippet: AMA effects on the production of type I IFNs and phosphorylation of IRF7 in TLR9-activated GEN2.2 cells. Cells were stimulated with the TLR9 agonist CpG-A (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A). The level of secreted IFN-α cytokine was determined by ELISA (B). Cells were stimulated for 30 min as described above then the levels of the phosphorylated (p-IRF7) and native form of IRF7 were determined by Western blotting (C, D). Bars represent the means ± SD of three individual experiments (A, B, D) and a representative blot is shown (C). ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05, ## p < 0.01, #### p < 0.0001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

    Dose- and time-dependent induction of cytosolic RIG-I receptor in GEN2.2 cells and the expression of RIG-I in AMA-exposed cells . In order to induce the cytosolic expression of RIG-I GEN2.2 cells were treated with increasing concentration of the specific TLR9 ligand, CpG-A (ranging from 0.01 to 0.5 μM). After 16 h the presence of RIG-I was detected in the cell lysates by Western blotting (A, B). To evaluate the time-dependent induction of RIG-I, GEN2.2 cells were exposed to 0.25 μM of CpG-A then the expression of RIG-I was measured in different time points (C, D). Finally cells were exposed to 0.25 μM of CpG-A in the presence or absence of AMA (0.5 μg/ml) or left untreated, for 16 h. Then cells were lysed and the protein level of RIG-I was assessed (E, F). Representative blots (A, C, E) and the means ± SD of four independent experiments (B, D, F) are shown. ***p < 0.001, ****p < 0.0001 vs. controls, ### p < 0.001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.
    Figure Legend Snippet: Dose- and time-dependent induction of cytosolic RIG-I receptor in GEN2.2 cells and the expression of RIG-I in AMA-exposed cells . In order to induce the cytosolic expression of RIG-I GEN2.2 cells were treated with increasing concentration of the specific TLR9 ligand, CpG-A (ranging from 0.01 to 0.5 μM). After 16 h the presence of RIG-I was detected in the cell lysates by Western blotting (A, B). To evaluate the time-dependent induction of RIG-I, GEN2.2 cells were exposed to 0.25 μM of CpG-A then the expression of RIG-I was measured in different time points (C, D). Finally cells were exposed to 0.25 μM of CpG-A in the presence or absence of AMA (0.5 μg/ml) or left untreated, for 16 h. Then cells were lysed and the protein level of RIG-I was assessed (E, F). Representative blots (A, C, E) and the means ± SD of four independent experiments (B, D, F) are shown. ***p < 0.001, ****p < 0.0001 vs. controls, ### p < 0.001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.

    Techniques Used: Expressing, Concentration Assay, Western Blot

    RIG-I and TLR9 induced type I IFN production in CpG-A-pre-conditioned GEN2.2 cells in the presence of AMA. Cells were pre-treated with CpG-A (0.25 μM) for 16 h to induce the cytosolic expression of RIG-I. Following thorough washing steps cells were re-exposed to the specific RIG-I ligand 5′ppp-dsRNA (RIGL, 1 μg/ml) in the presence or absence of AMA (0.5 μg/ml). The IFNA1 mRNA expression level was determined by real-time PCR after 3 h (A) and IFN-α protein level was assessed by ELISA after 6 h (B). In parallel experiments the re-activation of the cells was carried out with high dose of CpG-A (1 μM) in combination or not with AMA (0.5 μg/ml). IFNA1 mRNA (C) and IFN-α protein levels (D) were measured as in (A) and (B), respectively. Data are presented as means ± SD of four individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05 vs. RIGL and ## p < 0.01 vs. CpG-A. AMA: Antimycin-A, ND: not determined.
    Figure Legend Snippet: RIG-I and TLR9 induced type I IFN production in CpG-A-pre-conditioned GEN2.2 cells in the presence of AMA. Cells were pre-treated with CpG-A (0.25 μM) for 16 h to induce the cytosolic expression of RIG-I. Following thorough washing steps cells were re-exposed to the specific RIG-I ligand 5′ppp-dsRNA (RIGL, 1 μg/ml) in the presence or absence of AMA (0.5 μg/ml). The IFNA1 mRNA expression level was determined by real-time PCR after 3 h (A) and IFN-α protein level was assessed by ELISA after 6 h (B). In parallel experiments the re-activation of the cells was carried out with high dose of CpG-A (1 μM) in combination or not with AMA (0.5 μg/ml). IFNA1 mRNA (C) and IFN-α protein levels (D) were measured as in (A) and (B), respectively. Data are presented as means ± SD of four individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05 vs. RIGL and ## p < 0.01 vs. CpG-A. AMA: Antimycin-A, ND: not determined.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Activation Assay

    Effects of AMA treatments on the first and second waves of type I IFN production in primary human pDCs. Freshly isolated primary pDCs were stimulated with the TLR9 agonist CpG-A (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A) and the level of secreted IFN-α cytokine was determined by ELISA (B). To investigate the effects of AMA on the second wave of type I IFN responses, primary pDCs were pre-treated with CpG-A for 16 h then after thorough washing steps cells were re-exposed to 5′ppp-dsRNA (RIGL, 1 μg/ml) in the presence or absence of AMA (0.5 μg/ml). After 3 h the IFNA1 mRNA expression level was determined by real-time PCR (C) and after 6 h IFN-α protein level was assessed by ELISA (D). Data are presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05 vs. CpG-A or RIGL and ## p < 0.01 vs. CpG-A or RIGL. AMA: Antimycin-A, ND: not determined.
    Figure Legend Snippet: Effects of AMA treatments on the first and second waves of type I IFN production in primary human pDCs. Freshly isolated primary pDCs were stimulated with the TLR9 agonist CpG-A (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A) and the level of secreted IFN-α cytokine was determined by ELISA (B). To investigate the effects of AMA on the second wave of type I IFN responses, primary pDCs were pre-treated with CpG-A for 16 h then after thorough washing steps cells were re-exposed to 5′ppp-dsRNA (RIGL, 1 μg/ml) in the presence or absence of AMA (0.5 μg/ml). After 3 h the IFNA1 mRNA expression level was determined by real-time PCR (C) and after 6 h IFN-α protein level was assessed by ELISA (D). Data are presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05 vs. CpG-A or RIGL and ## p < 0.01 vs. CpG-A or RIGL. AMA: Antimycin-A, ND: not determined.

    Techniques Used: Isolation, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

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    Hycult Biotech tlr9 agonist cpg dna
    Tlr9 Agonist Cpg Dna, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hycult Biotech tlr9 agonist cpg a
    AMA effects on the production of type I IFNs and phosphorylation of IRF7 in <t>TLR9-activated</t> GEN2.2 cells. Cells were stimulated with the TLR9 agonist <t>CpG-A</t> (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A). The level of secreted IFN-α cytokine was determined by ELISA (B). Cells were stimulated for 30 min as described above then the levels of the phosphorylated (p-IRF7) and native form of IRF7 were determined by Western blotting (C, D). Bars represent the means ± SD of three individual experiments (A, B, D) and a representative blot is shown (C). ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05, ## p < 0.01, #### p < 0.0001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.
    Tlr9 Agonist Cpg A, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AMA effects on the production of type I IFNs and phosphorylation of IRF7 in TLR9-activated GEN2.2 cells. Cells were stimulated with the TLR9 agonist CpG-A (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A). The level of secreted IFN-α cytokine was determined by ELISA (B). Cells were stimulated for 30 min as described above then the levels of the phosphorylated (p-IRF7) and native form of IRF7 were determined by Western blotting (C, D). Bars represent the means ± SD of three individual experiments (A, B, D) and a representative blot is shown (C). ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05, ## p < 0.01, #### p < 0.0001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.

    Journal: Redox Biology

    Article Title: Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells

    doi: 10.1016/j.redox.2017.07.016

    Figure Lengend Snippet: AMA effects on the production of type I IFNs and phosphorylation of IRF7 in TLR9-activated GEN2.2 cells. Cells were stimulated with the TLR9 agonist CpG-A (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A). The level of secreted IFN-α cytokine was determined by ELISA (B). Cells were stimulated for 30 min as described above then the levels of the phosphorylated (p-IRF7) and native form of IRF7 were determined by Western blotting (C, D). Bars represent the means ± SD of three individual experiments (A, B, D) and a representative blot is shown (C). ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05, ## p < 0.01, #### p < 0.0001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.

    Article Snippet: For activation, cells were exposed to the TLR9 agonist CpG-A (ODN 2216, 1 μM; Cat. no. HC4037 from Hycult Biotech, Uden, The Netherlands) in the absence or presence of AMA (0.5 μg/ml; Sigma-Aldrich) for 6 h. The optimal concentration of AMA was chosen based on preliminary experiments performed on GEN2.2 cells.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

    Dose- and time-dependent induction of cytosolic RIG-I receptor in GEN2.2 cells and the expression of RIG-I in AMA-exposed cells . In order to induce the cytosolic expression of RIG-I GEN2.2 cells were treated with increasing concentration of the specific TLR9 ligand, CpG-A (ranging from 0.01 to 0.5 μM). After 16 h the presence of RIG-I was detected in the cell lysates by Western blotting (A, B). To evaluate the time-dependent induction of RIG-I, GEN2.2 cells were exposed to 0.25 μM of CpG-A then the expression of RIG-I was measured in different time points (C, D). Finally cells were exposed to 0.25 μM of CpG-A in the presence or absence of AMA (0.5 μg/ml) or left untreated, for 16 h. Then cells were lysed and the protein level of RIG-I was assessed (E, F). Representative blots (A, C, E) and the means ± SD of four independent experiments (B, D, F) are shown. ***p < 0.001, ****p < 0.0001 vs. controls, ### p < 0.001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.

    Journal: Redox Biology

    Article Title: Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells

    doi: 10.1016/j.redox.2017.07.016

    Figure Lengend Snippet: Dose- and time-dependent induction of cytosolic RIG-I receptor in GEN2.2 cells and the expression of RIG-I in AMA-exposed cells . In order to induce the cytosolic expression of RIG-I GEN2.2 cells were treated with increasing concentration of the specific TLR9 ligand, CpG-A (ranging from 0.01 to 0.5 μM). After 16 h the presence of RIG-I was detected in the cell lysates by Western blotting (A, B). To evaluate the time-dependent induction of RIG-I, GEN2.2 cells were exposed to 0.25 μM of CpG-A then the expression of RIG-I was measured in different time points (C, D). Finally cells were exposed to 0.25 μM of CpG-A in the presence or absence of AMA (0.5 μg/ml) or left untreated, for 16 h. Then cells were lysed and the protein level of RIG-I was assessed (E, F). Representative blots (A, C, E) and the means ± SD of four independent experiments (B, D, F) are shown. ***p < 0.001, ****p < 0.0001 vs. controls, ### p < 0.001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.

    Article Snippet: For activation, cells were exposed to the TLR9 agonist CpG-A (ODN 2216, 1 μM; Cat. no. HC4037 from Hycult Biotech, Uden, The Netherlands) in the absence or presence of AMA (0.5 μg/ml; Sigma-Aldrich) for 6 h. The optimal concentration of AMA was chosen based on preliminary experiments performed on GEN2.2 cells.

    Techniques: Expressing, Concentration Assay, Western Blot

    RIG-I and TLR9 induced type I IFN production in CpG-A-pre-conditioned GEN2.2 cells in the presence of AMA. Cells were pre-treated with CpG-A (0.25 μM) for 16 h to induce the cytosolic expression of RIG-I. Following thorough washing steps cells were re-exposed to the specific RIG-I ligand 5′ppp-dsRNA (RIGL, 1 μg/ml) in the presence or absence of AMA (0.5 μg/ml). The IFNA1 mRNA expression level was determined by real-time PCR after 3 h (A) and IFN-α protein level was assessed by ELISA after 6 h (B). In parallel experiments the re-activation of the cells was carried out with high dose of CpG-A (1 μM) in combination or not with AMA (0.5 μg/ml). IFNA1 mRNA (C) and IFN-α protein levels (D) were measured as in (A) and (B), respectively. Data are presented as means ± SD of four individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05 vs. RIGL and ## p < 0.01 vs. CpG-A. AMA: Antimycin-A, ND: not determined.

    Journal: Redox Biology

    Article Title: Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells

    doi: 10.1016/j.redox.2017.07.016

    Figure Lengend Snippet: RIG-I and TLR9 induced type I IFN production in CpG-A-pre-conditioned GEN2.2 cells in the presence of AMA. Cells were pre-treated with CpG-A (0.25 μM) for 16 h to induce the cytosolic expression of RIG-I. Following thorough washing steps cells were re-exposed to the specific RIG-I ligand 5′ppp-dsRNA (RIGL, 1 μg/ml) in the presence or absence of AMA (0.5 μg/ml). The IFNA1 mRNA expression level was determined by real-time PCR after 3 h (A) and IFN-α protein level was assessed by ELISA after 6 h (B). In parallel experiments the re-activation of the cells was carried out with high dose of CpG-A (1 μM) in combination or not with AMA (0.5 μg/ml). IFNA1 mRNA (C) and IFN-α protein levels (D) were measured as in (A) and (B), respectively. Data are presented as means ± SD of four individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05 vs. RIGL and ## p < 0.01 vs. CpG-A. AMA: Antimycin-A, ND: not determined.

    Article Snippet: For activation, cells were exposed to the TLR9 agonist CpG-A (ODN 2216, 1 μM; Cat. no. HC4037 from Hycult Biotech, Uden, The Netherlands) in the absence or presence of AMA (0.5 μg/ml; Sigma-Aldrich) for 6 h. The optimal concentration of AMA was chosen based on preliminary experiments performed on GEN2.2 cells.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Activation Assay

    Effects of AMA treatments on the first and second waves of type I IFN production in primary human pDCs. Freshly isolated primary pDCs were stimulated with the TLR9 agonist CpG-A (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A) and the level of secreted IFN-α cytokine was determined by ELISA (B). To investigate the effects of AMA on the second wave of type I IFN responses, primary pDCs were pre-treated with CpG-A for 16 h then after thorough washing steps cells were re-exposed to 5′ppp-dsRNA (RIGL, 1 μg/ml) in the presence or absence of AMA (0.5 μg/ml). After 3 h the IFNA1 mRNA expression level was determined by real-time PCR (C) and after 6 h IFN-α protein level was assessed by ELISA (D). Data are presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05 vs. CpG-A or RIGL and ## p < 0.01 vs. CpG-A or RIGL. AMA: Antimycin-A, ND: not determined.

    Journal: Redox Biology

    Article Title: Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells

    doi: 10.1016/j.redox.2017.07.016

    Figure Lengend Snippet: Effects of AMA treatments on the first and second waves of type I IFN production in primary human pDCs. Freshly isolated primary pDCs were stimulated with the TLR9 agonist CpG-A (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A) and the level of secreted IFN-α cytokine was determined by ELISA (B). To investigate the effects of AMA on the second wave of type I IFN responses, primary pDCs were pre-treated with CpG-A for 16 h then after thorough washing steps cells were re-exposed to 5′ppp-dsRNA (RIGL, 1 μg/ml) in the presence or absence of AMA (0.5 μg/ml). After 3 h the IFNA1 mRNA expression level was determined by real-time PCR (C) and after 6 h IFN-α protein level was assessed by ELISA (D). Data are presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control, # p < 0.05 vs. CpG-A or RIGL and ## p < 0.01 vs. CpG-A or RIGL. AMA: Antimycin-A, ND: not determined.

    Article Snippet: For activation, cells were exposed to the TLR9 agonist CpG-A (ODN 2216, 1 μM; Cat. no. HC4037 from Hycult Biotech, Uden, The Netherlands) in the absence or presence of AMA (0.5 μg/ml; Sigma-Aldrich) for 6 h. The optimal concentration of AMA was chosen based on preliminary experiments performed on GEN2.2 cells.

    Techniques: Isolation, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay