irf8-mediated inhibition Search Results


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  • 97
    Millipore ezh2
    <t>EZH2</t> directly silences IRF8 expression
    Ezh2, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 911 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc irf1
    <t>IRF1</t> functions in both the myeloid and epithelial cell to prevent colitis-associated colorectal tumorigenesis. ( A ) Representative images of colon tumors in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after azoxymethane (AOM) injection. ( B ) Number of colon tumors in WT ( n = 10), LysM Cre Irf1 fl/fl ( n = 10), Villin Cre Irf1 fl/fl ( n = 10), and Irf1 –/– ( n = 7) mice. ( C ) Percentage of tumors of various sizes in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after AOM injection. ( D ) Body weight change in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after AOM injection. Each symbol represents 1 individual mouse in B . * P
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    Cell Signaling Technology Inc akt
    Effects of wortmannin on NF- κ B and <t>IRF-3</t> nuclear translocation, I κ B degradation and <t>Akt</t> phosphorylation in H1N1-infected A549 cells. For nuclear translocation and cytosolic I κ B degradation, cells were lysed at 16 h p.i. and then protein was extracted for western blot analysis as described in Section 2 . While assessing Akt phosphorylation, cells were lysed at 6 h p.i. then cytosolic protein was extracted. Similar results were obtained in three independent experiments.
    Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 48620 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Unigene irf8
    Western blot analysis of NCoA3 and <t>IRF8</t> proteins expression . Nuclear extract (100 μg) from U1, ACH-2, J1.1 and OM10.1 treated (+) or not (-) with NaB for 24 h were resolved by SDS-PAGE and immunoblotted with anti-NCoA3 or anti-IRF8 antibody, as indicated. The amount of protein was normalized using anti-actin antibody. Figures below NCoA3 immunoblot indicated the results of the quantification using Image Tool (Syngene) software of the ratio NCoA3/actin upon NaB-treatment (+) versus NCoA3/actin non-treated (-). Results are representative of three independent experiments.
    Irf8, supplied by Unigene, used in various techniques. Bioz Stars score: 91/100, based on 127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc nlrx1
    <t>NLRX1</t> acts independently of ROS response to promote H. capsulatum -induced LC3 conversion in macrophages. (A,B,F) Macrophages from WT and Nlrx1 −/− mice were stimulated with or without (0 min) zymosan (50 μg/ml) (A) or H. capsulatum (MOI = 5) (B,F) for indicated time. Cell lysates were extracted and analyzed by Western blotting for the expression of indicated proteins. Data shown in the lower panel are relative intensity of LC3-II normalized against the corresponding β-actin, mean ± SEM are shown ( n = 3) (A,B) . (C,D) Macrophages from WT and Nlrx1 −/− mice were stimulated with H. capsulatum for 1 h. Cell were fixed and stained for LC3B (green), F-actin (red), and nucleus compartment (blue), and viewed under confocal microscope (C) . Asterisks in the DIC/Nucleus field point to H. capsulatum yeasts. The intensity of different fluorochromes along the white arrow in the merged image is shown as the histogram on the right. The mean fluorescence intensity (MFI) of LC3 in cells engulfing H. capsulatum was quantified. Phagosomes in each cell were counted and the percentages of LC3 + phagosome are shown as mean ± SEM of 3 independent experiments (D) . (E) Macrophages from WT and Nlrx1 −/− mice were allowed to phagocytose FITC-labeled H. capsulatum (MOI = 10) for 1 h. Percentages of cells engulfing H. capsulatum were analyzed by flow cytometry ( n = 7). (G) Macrophages from WT and Nlrx1 −/− mice were preloaded with CM-H 2 DCFDA for 30 min prior to stimulation with H. capsulatum (MOI = 10) for 20 min. Flow cytometry was performed to assess global ROS production. Data shown are the percentages of ROS + cells (left panel) and the mean fluorescence intensity (MFI) (right panel) of total live cells ( n = 4). Bars represent the mean ± SEM. ** p ≤ 0.01, *** p ≤ 0.001 [ANOVA with Bonferroni's multiple comparisons post-hoc test (A,B, G) ; 2-tailed t -test (D,E) ].
    Nlrx1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho akt
    <t>NLRX1</t> acts independently of ROS response to promote H. capsulatum -induced LC3 conversion in macrophages. (A,B,F) Macrophages from WT and Nlrx1 −/− mice were stimulated with or without (0 min) zymosan (50 μg/ml) (A) or H. capsulatum (MOI = 5) (B,F) for indicated time. Cell lysates were extracted and analyzed by Western blotting for the expression of indicated proteins. Data shown in the lower panel are relative intensity of LC3-II normalized against the corresponding β-actin, mean ± SEM are shown ( n = 3) (A,B) . (C,D) Macrophages from WT and Nlrx1 −/− mice were stimulated with H. capsulatum for 1 h. Cell were fixed and stained for LC3B (green), F-actin (red), and nucleus compartment (blue), and viewed under confocal microscope (C) . Asterisks in the DIC/Nucleus field point to H. capsulatum yeasts. The intensity of different fluorochromes along the white arrow in the merged image is shown as the histogram on the right. The mean fluorescence intensity (MFI) of LC3 in cells engulfing H. capsulatum was quantified. Phagosomes in each cell were counted and the percentages of LC3 + phagosome are shown as mean ± SEM of 3 independent experiments (D) . (E) Macrophages from WT and Nlrx1 −/− mice were allowed to phagocytose FITC-labeled H. capsulatum (MOI = 10) for 1 h. Percentages of cells engulfing H. capsulatum were analyzed by flow cytometry ( n = 7). (G) Macrophages from WT and Nlrx1 −/− mice were preloaded with CM-H 2 DCFDA for 30 min prior to stimulation with H. capsulatum (MOI = 10) for 20 min. Flow cytometry was performed to assess global ROS production. Data shown are the percentages of ROS + cells (left panel) and the mean fluorescence intensity (MFI) (right panel) of total live cells ( n = 4). Bars represent the mean ± SEM. ** p ≤ 0.01, *** p ≤ 0.001 [ANOVA with Bonferroni's multiple comparisons post-hoc test (A,B, G) ; 2-tailed t -test (D,E) ].
    Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 16290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc caspase 8
    <t>Caspase-8</t> is required for the proteasome-mediated degradation of IRF-3. A , HT1080 cells were transfected with poly(I:C) in the absence or the presence of an inhibitor of caspase-8 (z-IETD, 10 μ m ) for the indicated times, and cell lysates were
    Caspase 8, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 5126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc anti traf6
    TLR7 triggering leads to tyrosine kinase dependent Src K63-linked ubiquitination and TNFR-associated factor 6 <t>(TRAF6)</t> tyrosine phosphorylation. hTLR7-HEK293 cells, transiently transfected with plasmids coding for hemagglutinin (HA)-tagged K63-only, were pretreated or not with PP2 (20 µM) for 30 min and stimulated for 2 h with R848 (10 µM). (A) Total cell lysates were immunoprecipitated with anti-pSFKs antibody and immunoblotted using anti-HA antibody for ubiquitin detection. After stripping, the presence of p-SFKs was assessed by immunoblot analysis using specific antibodies, as control. (B) Total cell lysates were immunoprecipitated with anti-TRAF6 and immunoblotted with antibodies against pSFKs, Src, and pTyr. (C) TRAF6-specific immunoprecipitates were eluted with 2% SDS in order to disrupt the TRAF6-SFKs interaction and after dilution with PBS re-immunoprecipitated with anti-TRAF6 antibody. Results are representative of three independent experiments. Histograms on the right side of the blots show the quantification of the pulled-down proteins, calculated at least on two independent experiments. Each signal was normalized to the signal of the protein used for the pull-down and expressed as fold induction (mean ± SD) compared to untreated sample. *** P
    Anti Traf6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti foxo1
    A positive feedback loop comprised of <t>Foxo1-BLNK-p38</t> that regulates pre-B cell differentiation
    Anti Foxo1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1047 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p53
    SAHA-induced acetylation of <t>p53</t> led to dissociation from MDM2, contributing to cell apoptosis.
    Phospho P53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho rip1 s166
    SAHA-induced acetylation of <t>p53</t> led to dissociation from MDM2, contributing to cell apoptosis.
    Phospho Rip1 S166, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc tlr4
    <t>TLR4</t> is a direct target of miR-140-5p. (A) Schematic demonstration of the potential binding sites (Binding site 1 was conserved, while binding site 2 and 3 were poorly conserved) for miR-140-5p in the 3′UTR of TLR4. (B) Dual luciferase reporter assay. pmirGLO-TLR4-3′UTR-1 contained binding site 1 and pmirGLO-TLR4-3′UTR-2 contained binding sites 2 and 3. TLR4 protein expression due to the (C) overexpression and (D) inhibition of miR-140-5p was analyzed in BEAS-2B cells. *P
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    Durect Corporation 1002 osmotic minipump
    <t>TLR4</t> is a direct target of miR-140-5p. (A) Schematic demonstration of the potential binding sites (Binding site 1 was conserved, while binding site 2 and 3 were poorly conserved) for miR-140-5p in the 3′UTR of TLR4. (B) Dual luciferase reporter assay. pmirGLO-TLR4-3′UTR-1 contained binding site 1 and pmirGLO-TLR4-3′UTR-2 contained binding sites 2 and 3. TLR4 protein expression due to the (C) overexpression and (D) inhibition of miR-140-5p was analyzed in BEAS-2B cells. *P
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    Cell Signaling Technology Inc acetyl p53
    SAHA-induced acetylation of <t>p53</t> led to dissociation from MDM2, contributing to cell apoptosis.
    Acetyl P53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc akt pan
    SAHA-induced acetylation of <t>p53</t> led to dissociation from MDM2, contributing to cell apoptosis.
    Akt Pan, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 757 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc antibodies against pd l1
    SAHA-induced acetylation of <t>p53</t> led to dissociation from MDM2, contributing to cell apoptosis.
    Antibodies Against Pd L1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    EZH2 directly silences IRF8 expression

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

    Article Title: Cutting Edge: EZH2 promotes osteoclastogenesis by epigenetic silencing of the negative regulator IRF8 1

    doi: 10.4049/jimmunol.1501466

    Figure Lengend Snippet: EZH2 directly silences IRF8 expression

    Article Snippet: Collectively, these results show that EZH2 is recruited to IRF8 during the early stages of RANKL-induced osteoclast differentiation, and that EZH2-mediated H3K27me3 deposition represses IRF8 to facilitate osteoclastogenesis.

    Techniques: Expressing

    EZH2 is required for osteoclast differentiation and NFATc1 expression

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

    Article Title: Cutting Edge: EZH2 promotes osteoclastogenesis by epigenetic silencing of the negative regulator IRF8 1

    doi: 10.4049/jimmunol.1501466

    Figure Lengend Snippet: EZH2 is required for osteoclast differentiation and NFATc1 expression

    Article Snippet: Collectively, these results show that EZH2 is recruited to IRF8 during the early stages of RANKL-induced osteoclast differentiation, and that EZH2-mediated H3K27me3 deposition represses IRF8 to facilitate osteoclastogenesis.

    Techniques: Expressing

    IRF1 functions in both the myeloid and epithelial cell to prevent colitis-associated colorectal tumorigenesis. ( A ) Representative images of colon tumors in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after azoxymethane (AOM) injection. ( B ) Number of colon tumors in WT ( n = 10), LysM Cre Irf1 fl/fl ( n = 10), Villin Cre Irf1 fl/fl ( n = 10), and Irf1 –/– ( n = 7) mice. ( C ) Percentage of tumors of various sizes in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after AOM injection. ( D ) Body weight change in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after AOM injection. Each symbol represents 1 individual mouse in B . * P

    Journal: JCI Insight

    Article Title: Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer

    doi: 10.1172/jci.insight.136720

    Figure Lengend Snippet: IRF1 functions in both the myeloid and epithelial cell to prevent colitis-associated colorectal tumorigenesis. ( A ) Representative images of colon tumors in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after azoxymethane (AOM) injection. ( B ) Number of colon tumors in WT ( n = 10), LysM Cre Irf1 fl/fl ( n = 10), Villin Cre Irf1 fl/fl ( n = 10), and Irf1 –/– ( n = 7) mice. ( C ) Percentage of tumors of various sizes in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after AOM injection. ( D ) Body weight change in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after AOM injection. Each symbol represents 1 individual mouse in B . * P

    Article Snippet: Following electrophoretic transfer of proteins onto PVDF membranes (IPVH00010, MilliporeSigma), nonspecific binding was blocked by incubation with 5% skim milk, and then membranes were incubated with primary antibodies against CASP3 (9662, Cell Signaling Technology [CST]), cleaved CASP3 (9661, CST), CASP7 (9492, CST), cleaved CASP7 (9491, CST), CASP8 (AG-20T-0138-C100, AdipoGen), cleaved CASP8 (8592, CST), IRF1 (8478, CST), P-ERK (9101, CST), ERK (9102, CST), P-IκBα (9241, CST), IκBα (9242, CST), P-STAT3 Tyr705 (9131, CST), STAT3 (9139, CST), and GAPDH (5174, CST).

    Techniques: Mouse Assay, Injection

    IRF1 regulates PANoptosis. ( A ) Representative images of TUNEL staining of colons from azoxymethane/dextran sulfate sodium–treated (AOM/DSS-treated) WT and Irf1 –/– mice on days 0, 14, and 80. ( B ) Representative images of cleaved caspase-3 (CASP3) staining of colon tissues from DSS-treated WT and Irf1 –/– mice on days 0 and 14 after AOM injection. Scale bar: 100 μM. ( C ) Immunoblot analysis of the pro- and cleaved forms of CASP3 and caspase-7 (CASP7) in colons of WT and Irf1 –/– mice on days 0, 14, and 80 after AOM injection. Blots represent data from the same biological samples at the indicated time point run in parallel. ( D ) Immunoblot analysis of GSDMD and MLKL in colons of WT and Irf1 –/– mice 14 days after AOM injection. Blots represent data from the same biological samples run in parallel. ( E ) Cell death analysis of intestinal organoids derived from WT and Irf1 –/– mice after stimulation with TNF + zVAD. ** P

    Journal: JCI Insight

    Article Title: Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer

    doi: 10.1172/jci.insight.136720

    Figure Lengend Snippet: IRF1 regulates PANoptosis. ( A ) Representative images of TUNEL staining of colons from azoxymethane/dextran sulfate sodium–treated (AOM/DSS-treated) WT and Irf1 –/– mice on days 0, 14, and 80. ( B ) Representative images of cleaved caspase-3 (CASP3) staining of colon tissues from DSS-treated WT and Irf1 –/– mice on days 0 and 14 after AOM injection. Scale bar: 100 μM. ( C ) Immunoblot analysis of the pro- and cleaved forms of CASP3 and caspase-7 (CASP7) in colons of WT and Irf1 –/– mice on days 0, 14, and 80 after AOM injection. Blots represent data from the same biological samples at the indicated time point run in parallel. ( D ) Immunoblot analysis of GSDMD and MLKL in colons of WT and Irf1 –/– mice 14 days after AOM injection. Blots represent data from the same biological samples run in parallel. ( E ) Cell death analysis of intestinal organoids derived from WT and Irf1 –/– mice after stimulation with TNF + zVAD. ** P

    Article Snippet: Following electrophoretic transfer of proteins onto PVDF membranes (IPVH00010, MilliporeSigma), nonspecific binding was blocked by incubation with 5% skim milk, and then membranes were incubated with primary antibodies against CASP3 (9662, Cell Signaling Technology [CST]), cleaved CASP3 (9661, CST), CASP7 (9492, CST), cleaved CASP7 (9491, CST), CASP8 (AG-20T-0138-C100, AdipoGen), cleaved CASP8 (8592, CST), IRF1 (8478, CST), P-ERK (9101, CST), ERK (9102, CST), P-IκBα (9241, CST), IκBα (9242, CST), P-STAT3 Tyr705 (9131, CST), STAT3 (9139, CST), and GAPDH (5174, CST).

    Techniques: TUNEL Assay, Staining, Mouse Assay, Injection, Derivative Assay

    IRF1 prevents colitis-associated colorectal tumorigenesis. ( A ) Body weight change of WT ( n = 10) and Irf1 –/– ( n = 10) mice from 1 experiment (representative of 3 independent experiments). ( B ) Representative images of colon tumors in WT and Irf1 –/– mice 80 days after injection of azoxymethane (AOM). ( C ) Number of colon tumors in WT ( n = 14) and Irf1 –/– ( n = 12) mice. ( D ) Percentage of tumors of various sizes 80 days after AOM injection. ( E ) Representative H E staining of colon tumors. Scale bar: 200 μM. ( F ) Histological scores 80 days after injection of AOM. ( G ) Percentage of mice with dysplasia 80 days after AOM injection. ( H ) Percentage of mice with adenocarcinoma 80 days after AOM injection. Data are from 1 experiment (representative of 3 independent experiments). Each symbol represents 1 individual mouse ( C and F ). *** P

    Journal: JCI Insight

    Article Title: Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer

    doi: 10.1172/jci.insight.136720

    Figure Lengend Snippet: IRF1 prevents colitis-associated colorectal tumorigenesis. ( A ) Body weight change of WT ( n = 10) and Irf1 –/– ( n = 10) mice from 1 experiment (representative of 3 independent experiments). ( B ) Representative images of colon tumors in WT and Irf1 –/– mice 80 days after injection of azoxymethane (AOM). ( C ) Number of colon tumors in WT ( n = 14) and Irf1 –/– ( n = 12) mice. ( D ) Percentage of tumors of various sizes 80 days after AOM injection. ( E ) Representative H E staining of colon tumors. Scale bar: 200 μM. ( F ) Histological scores 80 days after injection of AOM. ( G ) Percentage of mice with dysplasia 80 days after AOM injection. ( H ) Percentage of mice with adenocarcinoma 80 days after AOM injection. Data are from 1 experiment (representative of 3 independent experiments). Each symbol represents 1 individual mouse ( C and F ). *** P

    Article Snippet: Following electrophoretic transfer of proteins onto PVDF membranes (IPVH00010, MilliporeSigma), nonspecific binding was blocked by incubation with 5% skim milk, and then membranes were incubated with primary antibodies against CASP3 (9662, Cell Signaling Technology [CST]), cleaved CASP3 (9661, CST), CASP7 (9492, CST), cleaved CASP7 (9491, CST), CASP8 (AG-20T-0138-C100, AdipoGen), cleaved CASP8 (8592, CST), IRF1 (8478, CST), P-ERK (9101, CST), ERK (9102, CST), P-IκBα (9241, CST), IκBα (9242, CST), P-STAT3 Tyr705 (9131, CST), STAT3 (9139, CST), and GAPDH (5174, CST).

    Techniques: Mouse Assay, Injection, Staining

    IRF1 prevents colorectal cancer in an Apc Min/+ model of tumorigenesis. ( A ) Representative images of colon tumors in 120-day-old littermate Apc Min/+ , Apc Min/+ Irf1 +/- , and Apc Min/+ Irf1 −/− mice. ( B ) Number of colon tumors in 120-day-old littermate Apc Min/+ ( n = 5), Apc Min/+ Irf1 +/– ( n = 5) , and Apc Min/+ Irf1 −/− ( n = 5) mice. ( C ) Percentage of tumors of various sizes in 120-day-old littermate Apc Min/+ , Apc Min/+ Irf1 +/– , and Apc Min/+ Irf1 −/− mice. Each symbol represents 1 individual mouse in B . ** P

    Journal: JCI Insight

    Article Title: Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer

    doi: 10.1172/jci.insight.136720

    Figure Lengend Snippet: IRF1 prevents colorectal cancer in an Apc Min/+ model of tumorigenesis. ( A ) Representative images of colon tumors in 120-day-old littermate Apc Min/+ , Apc Min/+ Irf1 +/- , and Apc Min/+ Irf1 −/− mice. ( B ) Number of colon tumors in 120-day-old littermate Apc Min/+ ( n = 5), Apc Min/+ Irf1 +/– ( n = 5) , and Apc Min/+ Irf1 −/− ( n = 5) mice. ( C ) Percentage of tumors of various sizes in 120-day-old littermate Apc Min/+ , Apc Min/+ Irf1 +/– , and Apc Min/+ Irf1 −/− mice. Each symbol represents 1 individual mouse in B . ** P

    Article Snippet: Following electrophoretic transfer of proteins onto PVDF membranes (IPVH00010, MilliporeSigma), nonspecific binding was blocked by incubation with 5% skim milk, and then membranes were incubated with primary antibodies against CASP3 (9662, Cell Signaling Technology [CST]), cleaved CASP3 (9661, CST), CASP7 (9492, CST), cleaved CASP7 (9491, CST), CASP8 (AG-20T-0138-C100, AdipoGen), cleaved CASP8 (8592, CST), IRF1 (8478, CST), P-ERK (9101, CST), ERK (9102, CST), P-IκBα (9241, CST), IκBα (9242, CST), P-STAT3 Tyr705 (9131, CST), STAT3 (9139, CST), and GAPDH (5174, CST).

    Techniques: Mouse Assay

    IRF1 does not regulate inflammation in the colon. ( A ) Body weight change of WT ( n = 10) and Irf1 –/– ( n = 10) mice from 1 experiment (representative of 3 independent experiments). ( B and C ) Representative images of colon ( B ) and length of colon ( C ) in WT ( n = 10) and Irf1 –/– ( n = 10) mice 14 days after azoxymethane (AOM) injection. ( D ) Histological scores. ( E ) Representative H E staining of colon. Scale bar: 500 μM. ( F ) Immunoblot analysis of IRF1, phosphorylated and total ERK1 and ERK2 (P-ERK1/2 and ERK1/2, respectively), phosphorylated and total IκBα (P-IκBα and IκBα, respectively), phosphorylated and total STAT3 (P-STAT3 and STAT3, respectively), and GAPDH (loading control) in colons of WT and Irf1 –/– mice. Blots represent data from the same biological samples run in parallel. ( G ) Levels of inflammatory cytokines in the colons of WT and Irf1 –/– mice at day 0, 14, and 80 after AOM injection. Each symbol represents 1 individual mouse ( C , D , and G ). * P

    Journal: JCI Insight

    Article Title: Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer

    doi: 10.1172/jci.insight.136720

    Figure Lengend Snippet: IRF1 does not regulate inflammation in the colon. ( A ) Body weight change of WT ( n = 10) and Irf1 –/– ( n = 10) mice from 1 experiment (representative of 3 independent experiments). ( B and C ) Representative images of colon ( B ) and length of colon ( C ) in WT ( n = 10) and Irf1 –/– ( n = 10) mice 14 days after azoxymethane (AOM) injection. ( D ) Histological scores. ( E ) Representative H E staining of colon. Scale bar: 500 μM. ( F ) Immunoblot analysis of IRF1, phosphorylated and total ERK1 and ERK2 (P-ERK1/2 and ERK1/2, respectively), phosphorylated and total IκBα (P-IκBα and IκBα, respectively), phosphorylated and total STAT3 (P-STAT3 and STAT3, respectively), and GAPDH (loading control) in colons of WT and Irf1 –/– mice. Blots represent data from the same biological samples run in parallel. ( G ) Levels of inflammatory cytokines in the colons of WT and Irf1 –/– mice at day 0, 14, and 80 after AOM injection. Each symbol represents 1 individual mouse ( C , D , and G ). * P

    Article Snippet: Following electrophoretic transfer of proteins onto PVDF membranes (IPVH00010, MilliporeSigma), nonspecific binding was blocked by incubation with 5% skim milk, and then membranes were incubated with primary antibodies against CASP3 (9662, Cell Signaling Technology [CST]), cleaved CASP3 (9661, CST), CASP7 (9492, CST), cleaved CASP7 (9491, CST), CASP8 (AG-20T-0138-C100, AdipoGen), cleaved CASP8 (8592, CST), IRF1 (8478, CST), P-ERK (9101, CST), ERK (9102, CST), P-IκBα (9241, CST), IκBα (9242, CST), P-STAT3 Tyr705 (9131, CST), STAT3 (9139, CST), and GAPDH (5174, CST).

    Techniques: Mouse Assay, Injection, Staining

    IRF1 suppresses the replication of HSV-1 partially by up-regulation of RSAD2. (A) HeLa cells were transfected with IRF1 and pcDNA3 or co-transfected with IRF1 and Pri-miR-23a and control vector, as indicated. Total RNA was extracted, and RSAD2 mRNA was quantified by quantitative real-time PCR. (B) HeLa cells were transfected with Myc-RSAD2. At 48-h post-transfection, quantitative real-time PCR was used to detect the level of RSAD2 mRNA, and at 72 h post-transfection, a Western blot was used to detect the expression level of RSAD2. (C) HeLa cells were transfected with Myc-RSAD2 or pcDNA3. Cells were infected with HSV-1 at 0.01 PFU/cell and stained with neutral red at 36 h post-infection. The mean radius of the cytopathic area was measured. The scale bar represents 100 µm. (D) HeLa cells were transfected with Myc-RSAD2 or pcDNA3. Viral yields were determined by standard plaque assays at 48 h post-infection with HSV-1. (E) Model of miR-23a regulation in HSV-1 replication. Increased levels of miR-23a in HeLa cells led to decrease levels of IRF1 mRNA and RSAD2 mRNA, with a consequent increase in HSV-1 replication. All data represent the mean value ± SD of at least three independent experiments. *: p

    Journal: PLoS ONE

    Article Title: MiR-23a Facilitates the Replication of HSV-1 through the Suppression of Interferon Regulatory Factor 1

    doi: 10.1371/journal.pone.0114021

    Figure Lengend Snippet: IRF1 suppresses the replication of HSV-1 partially by up-regulation of RSAD2. (A) HeLa cells were transfected with IRF1 and pcDNA3 or co-transfected with IRF1 and Pri-miR-23a and control vector, as indicated. Total RNA was extracted, and RSAD2 mRNA was quantified by quantitative real-time PCR. (B) HeLa cells were transfected with Myc-RSAD2. At 48-h post-transfection, quantitative real-time PCR was used to detect the level of RSAD2 mRNA, and at 72 h post-transfection, a Western blot was used to detect the expression level of RSAD2. (C) HeLa cells were transfected with Myc-RSAD2 or pcDNA3. Cells were infected with HSV-1 at 0.01 PFU/cell and stained with neutral red at 36 h post-infection. The mean radius of the cytopathic area was measured. The scale bar represents 100 µm. (D) HeLa cells were transfected with Myc-RSAD2 or pcDNA3. Viral yields were determined by standard plaque assays at 48 h post-infection with HSV-1. (E) Model of miR-23a regulation in HSV-1 replication. Increased levels of miR-23a in HeLa cells led to decrease levels of IRF1 mRNA and RSAD2 mRNA, with a consequent increase in HSV-1 replication. All data represent the mean value ± SD of at least three independent experiments. *: p

    Article Snippet: Following protein transfer to nitrocellulose membranes, the level of IRF1 expression was visualized by blotting with anti-IRF1 (Saier Biotech, Tianjin, China) and anti-FALG (Cell Signaling Technology, Beverly MA).

    Techniques: Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Infection, Staining

    IRF1 suppresses the replication of HSV-1. (A) HeLa cells were transfected with IRF1, sh-IRF1 and control vectors, respectively. Total RNA was extracted and analyzed for IRF1 mRNA by quantitative real-time PCR. The cell lysate was extracted and analyzed for IRF1 expression by Western blot. (B) HeLa cells were transfected as indicated in (A), MTT assay of cell viability was conducted at 24 h, 48 h and 72 h post-transfection. To up-regulate IRF1, two doses of vectors were used for transfection, 0.5 µg/well and 0.3 µg/well. Another group was transfected with sh-IRF1 and its control vector in the same way. (C–F) HeLa cells were transfected as indicated in (A), 24 h post-transfection, cells were infected with HSV-1 at 0.01 PFU/cell. At 48 h post-infection, cells were stained with neutral red. The mean radius of the cytopathic area was measured. The scale bar represents 100 µm (C). Total viral yields (D) and Yield of progeny virions from the culture supernatant (E) were determined by standard plaque assays. Level of glycoprotein expression was determined by immunofluorescence assay (F). All data represent the mean value ± SD of at least three independent experiments. *: p

    Journal: PLoS ONE

    Article Title: MiR-23a Facilitates the Replication of HSV-1 through the Suppression of Interferon Regulatory Factor 1

    doi: 10.1371/journal.pone.0114021

    Figure Lengend Snippet: IRF1 suppresses the replication of HSV-1. (A) HeLa cells were transfected with IRF1, sh-IRF1 and control vectors, respectively. Total RNA was extracted and analyzed for IRF1 mRNA by quantitative real-time PCR. The cell lysate was extracted and analyzed for IRF1 expression by Western blot. (B) HeLa cells were transfected as indicated in (A), MTT assay of cell viability was conducted at 24 h, 48 h and 72 h post-transfection. To up-regulate IRF1, two doses of vectors were used for transfection, 0.5 µg/well and 0.3 µg/well. Another group was transfected with sh-IRF1 and its control vector in the same way. (C–F) HeLa cells were transfected as indicated in (A), 24 h post-transfection, cells were infected with HSV-1 at 0.01 PFU/cell. At 48 h post-infection, cells were stained with neutral red. The mean radius of the cytopathic area was measured. The scale bar represents 100 µm (C). Total viral yields (D) and Yield of progeny virions from the culture supernatant (E) were determined by standard plaque assays. Level of glycoprotein expression was determined by immunofluorescence assay (F). All data represent the mean value ± SD of at least three independent experiments. *: p

    Article Snippet: Following protein transfer to nitrocellulose membranes, the level of IRF1 expression was visualized by blotting with anti-IRF1 (Saier Biotech, Tianjin, China) and anti-FALG (Cell Signaling Technology, Beverly MA).

    Techniques: Transfection, Real-time Polymerase Chain Reaction, Expressing, Western Blot, MTT Assay, Plasmid Preparation, Infection, Staining, Immunofluorescence

    IRF1 is the direct target of miR-23a. (A) As predicted in the TargetScan database, the IRF1 3′UTR carries a miR-23a-binding site. The IRF1 3′UTR mutant, containing four mutated nucleotides within the miR-23a-binding site, is shown. The mutated nucleotides are marked in red. (B) HeLa cells were transfected with pcDNA3, Pri-miR-23a, pRNAT-U6.2 and Anti-miR-23a respectively, and co-transfected with pcDNA3/EGFP-IRF1-UTR reporter vector or pcDNA3/EGFP-IRF1-MUT mutant vector, as indicated. At 48 h post-transfection, the cell lysate was prepared to measure the EGFP intensity and the fluorescence value in the control group was set to 1. (C) HeLa cells were transfected with Pri-miR-23a, pcDNA3, pRNAT-U6.2 and Anti-miR-23a, respectively. At 48 h post-transfection, RNA was extracted from transfected HeLa cells and the IRF1 mRNA was quantified by real-time PCR. GAPDH mRNA was used as an internal control, and the group control was the level of IRF1 mRNA. The protein level of IRF1 was measured by Western blot. All data represent the mean value ± SD of at least three independent experiments. *p

    Journal: PLoS ONE

    Article Title: MiR-23a Facilitates the Replication of HSV-1 through the Suppression of Interferon Regulatory Factor 1

    doi: 10.1371/journal.pone.0114021

    Figure Lengend Snippet: IRF1 is the direct target of miR-23a. (A) As predicted in the TargetScan database, the IRF1 3′UTR carries a miR-23a-binding site. The IRF1 3′UTR mutant, containing four mutated nucleotides within the miR-23a-binding site, is shown. The mutated nucleotides are marked in red. (B) HeLa cells were transfected with pcDNA3, Pri-miR-23a, pRNAT-U6.2 and Anti-miR-23a respectively, and co-transfected with pcDNA3/EGFP-IRF1-UTR reporter vector or pcDNA3/EGFP-IRF1-MUT mutant vector, as indicated. At 48 h post-transfection, the cell lysate was prepared to measure the EGFP intensity and the fluorescence value in the control group was set to 1. (C) HeLa cells were transfected with Pri-miR-23a, pcDNA3, pRNAT-U6.2 and Anti-miR-23a, respectively. At 48 h post-transfection, RNA was extracted from transfected HeLa cells and the IRF1 mRNA was quantified by real-time PCR. GAPDH mRNA was used as an internal control, and the group control was the level of IRF1 mRNA. The protein level of IRF1 was measured by Western blot. All data represent the mean value ± SD of at least three independent experiments. *p

    Article Snippet: Following protein transfer to nitrocellulose membranes, the level of IRF1 expression was visualized by blotting with anti-IRF1 (Saier Biotech, Tianjin, China) and anti-FALG (Cell Signaling Technology, Beverly MA).

    Techniques: Binding Assay, Mutagenesis, Transfection, Plasmid Preparation, Fluorescence, Real-time Polymerase Chain Reaction, Western Blot

    Time course of miR-23a and IRF1 in HeLa cells infected with HSV-1. (A) miR-23a expression was determined by quantitative real-time PCR at indicated time. Fold-increase is shown compared with HeLa cells at each time. (B) Time course of IRF1 expression in HeLa cells infected with HSV-1. Fold-increase is shown compared with HeLa cells at each time.

    Journal: PLoS ONE

    Article Title: MiR-23a Facilitates the Replication of HSV-1 through the Suppression of Interferon Regulatory Factor 1

    doi: 10.1371/journal.pone.0114021

    Figure Lengend Snippet: Time course of miR-23a and IRF1 in HeLa cells infected with HSV-1. (A) miR-23a expression was determined by quantitative real-time PCR at indicated time. Fold-increase is shown compared with HeLa cells at each time. (B) Time course of IRF1 expression in HeLa cells infected with HSV-1. Fold-increase is shown compared with HeLa cells at each time.

    Article Snippet: Following protein transfer to nitrocellulose membranes, the level of IRF1 expression was visualized by blotting with anti-IRF1 (Saier Biotech, Tianjin, China) and anti-FALG (Cell Signaling Technology, Beverly MA).

    Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction

    Transfection with IRF1 cDNA lacking a 3′UTR counteracts the effects of miR-23a on HSV-1 replication. (A) HeLa cells were co-transfected with two of pcDNA3, Pri-miR-23a and IRF1. At 72 h post-transfection, Western blot was used to detect the expression level of IRF1. (B) and (C), HeLa cells were transfected with either IRF1 or control vector, along with Pri-miR-23a or control vector, as indicated. At 24 h post-transfection, cells were infected with HSV-1 at 0.01 PFU/cell. Plaques were stained with neutral red, and viral yields were determined by standard plaque assays. Scale bar represents 100 µm. All data represent the mean value ± SD of at least three independent experiments. * p

    Journal: PLoS ONE

    Article Title: MiR-23a Facilitates the Replication of HSV-1 through the Suppression of Interferon Regulatory Factor 1

    doi: 10.1371/journal.pone.0114021

    Figure Lengend Snippet: Transfection with IRF1 cDNA lacking a 3′UTR counteracts the effects of miR-23a on HSV-1 replication. (A) HeLa cells were co-transfected with two of pcDNA3, Pri-miR-23a and IRF1. At 72 h post-transfection, Western blot was used to detect the expression level of IRF1. (B) and (C), HeLa cells were transfected with either IRF1 or control vector, along with Pri-miR-23a or control vector, as indicated. At 24 h post-transfection, cells were infected with HSV-1 at 0.01 PFU/cell. Plaques were stained with neutral red, and viral yields were determined by standard plaque assays. Scale bar represents 100 µm. All data represent the mean value ± SD of at least three independent experiments. * p

    Article Snippet: Following protein transfer to nitrocellulose membranes, the level of IRF1 expression was visualized by blotting with anti-IRF1 (Saier Biotech, Tianjin, China) and anti-FALG (Cell Signaling Technology, Beverly MA).

    Techniques: Transfection, Western Blot, Expressing, Plasmid Preparation, Infection, Staining

    Value of Ki67 and IRF-1 in predicting post LT HCC recurrence ( A ) Statistically significant differences among all the patients were obtained for RFS between negative and positive groups of Ki67 ( P = 1.6 × 10 −4 , Bonferroni correction α′ = 1.5 × 10 −3 ). ( B ) Difference in RFS between negative and positive groups of Ki67 in the patients with T1-T3a HCC ( P = 6.8 × 10 −4 ). ( C ) A significant correlation was obtained between Ki-67 and T stage in the primary, but not recurrent, HCC group (Spearman correlation R = 0.459, p = 1.2 × 10 −5 and R = −0.139, P = 0.527). *: Extreme outliers. ( D ) A significant negative correlation was obtained between IRF-1 and Ki-67 (Spearman correlation R = −0.405, P = 0.030). ○: Mild outliers. ( E ) Among all the patients, differences in RFSs between negative and positive groups of IRF-1 failed to achieve statistical significance after Bonferroni correction ( P = 0.023, Bonferroni correction α′ = 1.5 × 10 −3 ). ( F ) In patients with HCCs beyond the Milan criteria, a significant difference in RFS was found between the negative and positive groups of IRF-1 ( P = 6.4 × 10 −5 , Bonferroni correction α′ = 1.5 × 10 −3 ).

    Journal: Oncotarget

    Article Title: Bi-directional roles of IRF-1 on autophagy diminish its prognostic value as compared with Ki67 in liver transplantation for hepatocellular carcinoma

    doi: 10.18632/oncotarget.9365

    Figure Lengend Snippet: Value of Ki67 and IRF-1 in predicting post LT HCC recurrence ( A ) Statistically significant differences among all the patients were obtained for RFS between negative and positive groups of Ki67 ( P = 1.6 × 10 −4 , Bonferroni correction α′ = 1.5 × 10 −3 ). ( B ) Difference in RFS between negative and positive groups of Ki67 in the patients with T1-T3a HCC ( P = 6.8 × 10 −4 ). ( C ) A significant correlation was obtained between Ki-67 and T stage in the primary, but not recurrent, HCC group (Spearman correlation R = 0.459, p = 1.2 × 10 −5 and R = −0.139, P = 0.527). *: Extreme outliers. ( D ) A significant negative correlation was obtained between IRF-1 and Ki-67 (Spearman correlation R = −0.405, P = 0.030). ○: Mild outliers. ( E ) Among all the patients, differences in RFSs between negative and positive groups of IRF-1 failed to achieve statistical significance after Bonferroni correction ( P = 0.023, Bonferroni correction α′ = 1.5 × 10 −3 ). ( F ) In patients with HCCs beyond the Milan criteria, a significant difference in RFS was found between the negative and positive groups of IRF-1 ( P = 6.4 × 10 −5 , Bonferroni correction α′ = 1.5 × 10 −3 ).

    Article Snippet: After proteins were transferred to polyvinylidene fluoride membranes (Millipore), each protein was detected with its antibody, including IRF-1 (1:1500, CST) and LC3A/B antibodies (1:1500, CST). β-actin was used as an internal control.

    Techniques:

    IFN-γ suppressed autophagy via caspase activation in SK-Hep1 cells ( A ) With the addition of Z-VAD-FMK, levels of cleaved Caspase-3 and PARP1 were decreased. ( B ) Levels of Beclin1, Atg5, Atg7 and LC3-II were decreased after IFN-γ stimulation. However, when IFN-γ was combined with the caspase inhibitor, levels of Beclin1, Atg5, Atg7 and LC3-II were not decreased as determined by the Western Blot test. ( C ) Summary of the roles of IRF-1 in apoptosis and autophagy.

    Journal: Oncotarget

    Article Title: Bi-directional roles of IRF-1 on autophagy diminish its prognostic value as compared with Ki67 in liver transplantation for hepatocellular carcinoma

    doi: 10.18632/oncotarget.9365

    Figure Lengend Snippet: IFN-γ suppressed autophagy via caspase activation in SK-Hep1 cells ( A ) With the addition of Z-VAD-FMK, levels of cleaved Caspase-3 and PARP1 were decreased. ( B ) Levels of Beclin1, Atg5, Atg7 and LC3-II were decreased after IFN-γ stimulation. However, when IFN-γ was combined with the caspase inhibitor, levels of Beclin1, Atg5, Atg7 and LC3-II were not decreased as determined by the Western Blot test. ( C ) Summary of the roles of IRF-1 in apoptosis and autophagy.

    Article Snippet: After proteins were transferred to polyvinylidene fluoride membranes (Millipore), each protein was detected with its antibody, including IRF-1 (1:1500, CST) and LC3A/B antibodies (1:1500, CST). β-actin was used as an internal control.

    Techniques: Activation Assay, Western Blot

    IFN-γ induced apoptosis in SK-Hep1 cells is associated with increasing levels of IRF-1 and pSTAT1 ( A ) Viability of living cells was decreased as a function of time in the IFN-γ group (mean ± SD; * P

    Journal: Oncotarget

    Article Title: Bi-directional roles of IRF-1 on autophagy diminish its prognostic value as compared with Ki67 in liver transplantation for hepatocellular carcinoma

    doi: 10.18632/oncotarget.9365

    Figure Lengend Snippet: IFN-γ induced apoptosis in SK-Hep1 cells is associated with increasing levels of IRF-1 and pSTAT1 ( A ) Viability of living cells was decreased as a function of time in the IFN-γ group (mean ± SD; * P

    Article Snippet: After proteins were transferred to polyvinylidene fluoride membranes (Millipore), each protein was detected with its antibody, including IRF-1 (1:1500, CST) and LC3A/B antibodies (1:1500, CST). β-actin was used as an internal control.

    Techniques:

    IFN-γ suppressed autophagy via IRF-1 in SK-Hep1 cells ( A ) Levels of LC3-II and Beclin1 were decreased in SK-Hep1 cells stimulated with IFN-γ. ( B ) and ( C ) In GFP-RFP-LC3 transfected SK-Hep1 cells, there was a decrease in the number of fluorescent spots after IFN-γ stimulation ( P

    Journal: Oncotarget

    Article Title: Bi-directional roles of IRF-1 on autophagy diminish its prognostic value as compared with Ki67 in liver transplantation for hepatocellular carcinoma

    doi: 10.18632/oncotarget.9365

    Figure Lengend Snippet: IFN-γ suppressed autophagy via IRF-1 in SK-Hep1 cells ( A ) Levels of LC3-II and Beclin1 were decreased in SK-Hep1 cells stimulated with IFN-γ. ( B ) and ( C ) In GFP-RFP-LC3 transfected SK-Hep1 cells, there was a decrease in the number of fluorescent spots after IFN-γ stimulation ( P

    Article Snippet: After proteins were transferred to polyvinylidene fluoride membranes (Millipore), each protein was detected with its antibody, including IRF-1 (1:1500, CST) and LC3A/B antibodies (1:1500, CST). β-actin was used as an internal control.

    Techniques: Transfection

    K-bZIP is able to SUMOylate IRF-1 and IRF-2 and to interact with IRF-7. Flag-tagged IRF-1 (A) , IRF-2 (B) and IRF-7 (C) were transiently co-transfected with the indicated tagged constructs. Total cell lysates (TCLs) were prepared 48 hours after transfection. The lysates were immunoprecipitated with M2-beads and analyzed by immunoblotting using anti-IRF-1 ( A ; right panel), anti-IRF-2 ( B ; right panel), anti-SUMO-2/3 ( A and B ; left panel) and anti-IRF-7 (C) antibodies. (D) TCLs from TREx-F3H3-K-bZIP BCBL-1 cells before and after 48 hours of Dox induction for K-bZIP overexpression were used for immunoprecipitation with anti-K-bZIP antibody. The immunoprecipitates and TCLs were analyzed by immunoblot with antibodies as indicated.

    Journal: BMC Genomics

    Article Title: The chromatin modification by SUMO-2/3 but not SUMO-1 prevents the epigenetic activation of key immune-related genes during Kaposi's sarcoma associated herpesvirus reactivation

    doi: 10.1186/1471-2164-14-824

    Figure Lengend Snippet: K-bZIP is able to SUMOylate IRF-1 and IRF-2 and to interact with IRF-7. Flag-tagged IRF-1 (A) , IRF-2 (B) and IRF-7 (C) were transiently co-transfected with the indicated tagged constructs. Total cell lysates (TCLs) were prepared 48 hours after transfection. The lysates were immunoprecipitated with M2-beads and analyzed by immunoblotting using anti-IRF-1 ( A ; right panel), anti-IRF-2 ( B ; right panel), anti-SUMO-2/3 ( A and B ; left panel) and anti-IRF-7 (C) antibodies. (D) TCLs from TREx-F3H3-K-bZIP BCBL-1 cells before and after 48 hours of Dox induction for K-bZIP overexpression were used for immunoprecipitation with anti-K-bZIP antibody. The immunoprecipitates and TCLs were analyzed by immunoblot with antibodies as indicated.

    Article Snippet: Antibodies used for immunoblotting were anti-IRF-1 (Cell Signaling Technologies, Beverly, MA), anti-IRF-2 (Cell Signaling Technologies), anti-IRF-7 (GeneTex), anti-SUMO-2/3 antibodies.

    Techniques: Transfection, Construct, Immunoprecipitation, Over Expression

    Confirmation of data derived from ChIP-seq for IRF-1, IRF-2 and IRF-7 binding sites with SUMO-2/3 enrichment relevant to K-Rta induction of KSHV reactivation in BCBL-1 cells. Chromatin samples derived from K-Rta-inducible BCBL-1 cells before and after 12 hours of K-Rta induction were used in ChIP reactions with antibodies specific for SUMO-1 and SUMO-2/3. Following ChIP assay, the IRF binding sites within the promoters of the genes, which are indicated at the bottom of the figure, were amplified using qPCR. All reactions were run in triplicate and normalized against the input. Nonspecific IgG was used as the control ChIP antibody.

    Journal: BMC Genomics

    Article Title: The chromatin modification by SUMO-2/3 but not SUMO-1 prevents the epigenetic activation of key immune-related genes during Kaposi's sarcoma associated herpesvirus reactivation

    doi: 10.1186/1471-2164-14-824

    Figure Lengend Snippet: Confirmation of data derived from ChIP-seq for IRF-1, IRF-2 and IRF-7 binding sites with SUMO-2/3 enrichment relevant to K-Rta induction of KSHV reactivation in BCBL-1 cells. Chromatin samples derived from K-Rta-inducible BCBL-1 cells before and after 12 hours of K-Rta induction were used in ChIP reactions with antibodies specific for SUMO-1 and SUMO-2/3. Following ChIP assay, the IRF binding sites within the promoters of the genes, which are indicated at the bottom of the figure, were amplified using qPCR. All reactions were run in triplicate and normalized against the input. Nonspecific IgG was used as the control ChIP antibody.

    Article Snippet: Antibodies used for immunoblotting were anti-IRF-1 (Cell Signaling Technologies, Beverly, MA), anti-IRF-2 (Cell Signaling Technologies), anti-IRF-7 (GeneTex), anti-SUMO-2/3 antibodies.

    Techniques: Derivative Assay, Chromatin Immunoprecipitation, Binding Assay, Amplification, Real-time Polymerase Chain Reaction

    SUMO-2/3 enrichment in stabilizing gene transcriptional during KSHV reactivation. (A) TREx-F3H3-K-Rta-shSUMO-2/3 BCBL-1 cells were treated with Dox for 24 and 48 hours. TCLs were analyzed by immunoblotting using anti-SUMO-2/3 antibody. (B) Twelve IRF-1, IRF-2 and IRF-7 targeted genes showing SUMO-2/3 enrichment at the promoter region during KSHV reactivation were chosen. Two genes showing no SUMO-2/3 enrichment at the promoter region were chosen as control. RNA samples derived from TREx-F3H3-K-Rta BCBL-1 and TREx-F3H3-K-Rta-shSUMO-2/3 BCBL-1 cells before and after 24 hours of Dox induction were subjected to reverse transcription (RT) reaction. Following the RT reaction, the IRF target genes were amplified by qPCR using gene-specific primer sets. All reactions were run in triplicate and normalized against GAPDH.

    Journal: BMC Genomics

    Article Title: The chromatin modification by SUMO-2/3 but not SUMO-1 prevents the epigenetic activation of key immune-related genes during Kaposi's sarcoma associated herpesvirus reactivation

    doi: 10.1186/1471-2164-14-824

    Figure Lengend Snippet: SUMO-2/3 enrichment in stabilizing gene transcriptional during KSHV reactivation. (A) TREx-F3H3-K-Rta-shSUMO-2/3 BCBL-1 cells were treated with Dox for 24 and 48 hours. TCLs were analyzed by immunoblotting using anti-SUMO-2/3 antibody. (B) Twelve IRF-1, IRF-2 and IRF-7 targeted genes showing SUMO-2/3 enrichment at the promoter region during KSHV reactivation were chosen. Two genes showing no SUMO-2/3 enrichment at the promoter region were chosen as control. RNA samples derived from TREx-F3H3-K-Rta BCBL-1 and TREx-F3H3-K-Rta-shSUMO-2/3 BCBL-1 cells before and after 24 hours of Dox induction were subjected to reverse transcription (RT) reaction. Following the RT reaction, the IRF target genes were amplified by qPCR using gene-specific primer sets. All reactions were run in triplicate and normalized against GAPDH.

    Article Snippet: Antibodies used for immunoblotting were anti-IRF-1 (Cell Signaling Technologies, Beverly, MA), anti-IRF-2 (Cell Signaling Technologies), anti-IRF-7 (GeneTex), anti-SUMO-2/3 antibodies.

    Techniques: Derivative Assay, Amplification, Real-time Polymerase Chain Reaction

    Overview of SUMO enrichment at IRF-1, IRF-2 and IRF-7 binding sites during viral reactivation. Percentage of SUMO-1 and SUMO-2/3 target gene numbers with SUMO enrichment at IRF-1 (A) , IRF-2 (B) or IRF-7 (C) binding sites in promoter regions before and after K-Rta induction for KSHV reactivation; numbers indicate counts of overlapping and non-overlapping gene numbers.

    Journal: BMC Genomics

    Article Title: The chromatin modification by SUMO-2/3 but not SUMO-1 prevents the epigenetic activation of key immune-related genes during Kaposi's sarcoma associated herpesvirus reactivation

    doi: 10.1186/1471-2164-14-824

    Figure Lengend Snippet: Overview of SUMO enrichment at IRF-1, IRF-2 and IRF-7 binding sites during viral reactivation. Percentage of SUMO-1 and SUMO-2/3 target gene numbers with SUMO enrichment at IRF-1 (A) , IRF-2 (B) or IRF-7 (C) binding sites in promoter regions before and after K-Rta induction for KSHV reactivation; numbers indicate counts of overlapping and non-overlapping gene numbers.

    Article Snippet: Antibodies used for immunoblotting were anti-IRF-1 (Cell Signaling Technologies, Beverly, MA), anti-IRF-2 (Cell Signaling Technologies), anti-IRF-7 (GeneTex), anti-SUMO-2/3 antibodies.

    Techniques: Binding Assay

    Effect of EGFR signaling on IRF1-induced CXCL10 production. Top , respiratory viruses (e.g., influenza virus, RV, and RSV) stimulate airway epithelial NADPH oxidase (Nox), metalloproteinase (MP), and ligand-induced activation of EGFR, which leads to IL-8 production. EGFR activation suppresses IRF1-induced CXCL10. Bottom , in the presence of EGFR inhibition (e.g., gefitinib and AG-1478), IRF1-induced CXCL10 is increased.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: EGFR activation suppresses respiratory virus-induced IRF1-dependent CXCL10 production

    doi: 10.1152/ajplung.00368.2013

    Figure Lengend Snippet: Effect of EGFR signaling on IRF1-induced CXCL10 production. Top , respiratory viruses (e.g., influenza virus, RV, and RSV) stimulate airway epithelial NADPH oxidase (Nox), metalloproteinase (MP), and ligand-induced activation of EGFR, which leads to IL-8 production. EGFR activation suppresses IRF1-induced CXCL10. Bottom , in the presence of EGFR inhibition (e.g., gefitinib and AG-1478), IRF1-induced CXCL10 is increased.

    Article Snippet: After electrophoresis and blocking with TBST (Bio-Rad Laboratories) containing 5% BSA, blots were then incubated with anti-IRF1 Ab (D5E4; Cell Signaling) overnight.

    Techniques: Activation Assay, Inhibition

    EGFR signaling affects virus-induced perforin and NK cell migration. A : BEAS-2b cells were treated with serum-free medium alone or transfected with IRF1 or control (C) siRNA for 24 h and treated with serum-free medium alone (white bars), H1N1 (hatched bars), RV (black bars), and RSV (gray bars), or each virus + gefitinib (Gef; 10 μM). After viral infection (24 h), secreted CXCL10 was measured by ELISA ( n = 6 independent experiments, means ± SE; * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: EGFR activation suppresses respiratory virus-induced IRF1-dependent CXCL10 production

    doi: 10.1152/ajplung.00368.2013

    Figure Lengend Snippet: EGFR signaling affects virus-induced perforin and NK cell migration. A : BEAS-2b cells were treated with serum-free medium alone or transfected with IRF1 or control (C) siRNA for 24 h and treated with serum-free medium alone (white bars), H1N1 (hatched bars), RV (black bars), and RSV (gray bars), or each virus + gefitinib (Gef; 10 μM). After viral infection (24 h), secreted CXCL10 was measured by ELISA ( n = 6 independent experiments, means ± SE; * P

    Article Snippet: After electrophoresis and blocking with TBST (Bio-Rad Laboratories) containing 5% BSA, blots were then incubated with anti-IRF1 Ab (D5E4; Cell Signaling) overnight.

    Techniques: Migration, Transfection, Infection, Enzyme-linked Immunosorbent Assay

    EGFR activation suppresses interferon regulatory factor (IRF) 1-induced CXCL10 production. A : BEAS-2b cells were treated with serum-free medium alone, or transfected with IRF1 or control (C) siRNA for 24 h and treated with serum-free medium alone (white bars), or H1N1 (hatched bars), RV (black bars), and RSV (gray bars). After viral infection (24 h), secreted CXCL10 was measured by ELISA ( n = 5 independent experiments, means ± SE; * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: EGFR activation suppresses respiratory virus-induced IRF1-dependent CXCL10 production

    doi: 10.1152/ajplung.00368.2013

    Figure Lengend Snippet: EGFR activation suppresses interferon regulatory factor (IRF) 1-induced CXCL10 production. A : BEAS-2b cells were treated with serum-free medium alone, or transfected with IRF1 or control (C) siRNA for 24 h and treated with serum-free medium alone (white bars), or H1N1 (hatched bars), RV (black bars), and RSV (gray bars). After viral infection (24 h), secreted CXCL10 was measured by ELISA ( n = 5 independent experiments, means ± SE; * P

    Article Snippet: After electrophoresis and blocking with TBST (Bio-Rad Laboratories) containing 5% BSA, blots were then incubated with anti-IRF1 Ab (D5E4; Cell Signaling) overnight.

    Techniques: Activation Assay, Transfection, Infection, Enzyme-linked Immunosorbent Assay

    EGFR inhibition increases IRF1-induced CXCL10 production in vitro and in vivo. A : BEAS-2b cells were treated with serum-free medium alone (white bars), gefitinib (Gef; 10 μM), AG-1295 (10 μM), H1N1 (hatched bars), RV (black bars), and RSV (gray bars), or virus + Gef and AG-1295, and secreted CXCL10 protein was measured by ELISA at 24 h ( n = 3–6 independent experiments, means ± SE; * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: EGFR activation suppresses respiratory virus-induced IRF1-dependent CXCL10 production

    doi: 10.1152/ajplung.00368.2013

    Figure Lengend Snippet: EGFR inhibition increases IRF1-induced CXCL10 production in vitro and in vivo. A : BEAS-2b cells were treated with serum-free medium alone (white bars), gefitinib (Gef; 10 μM), AG-1295 (10 μM), H1N1 (hatched bars), RV (black bars), and RSV (gray bars), or virus + Gef and AG-1295, and secreted CXCL10 protein was measured by ELISA at 24 h ( n = 3–6 independent experiments, means ± SE; * P

    Article Snippet: After electrophoresis and blocking with TBST (Bio-Rad Laboratories) containing 5% BSA, blots were then incubated with anti-IRF1 Ab (D5E4; Cell Signaling) overnight.

    Techniques: Inhibition, In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay

    CMLD candidate compound identification and validation. ( a ) Dose response in J774-21 GFP-Ipr1 reporter cell line. J7-21 cells were treated with different doses of C9433 (0.010, 0.033, 0.1, 0.33, 1, 3.3 μM) in the presence of 0.2 U/mL IFNγ for 24 hrs, followed by addition of 1 μg/mL dox for 24 hrs. GFP-Ipr1 expression was measured using automated cytometry. Results are representative of at least two independent experiments performed in triplicates. ( b ) Toxicity of compounds in primary BMDM. BMDM were treated with compounds C9433, C5557 and C8808 at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. ( c ) Dose-dependent translation inhibition by rocaglates. 293TR-FLuc cells were treated with rocaglates C9433, C5557 and C8808 (0.033, 0.1, 0.33, 1, 3.3 μM) and luciferase activity was measured after 18 hrs. Two independent experiments were performed in triplicates. ( d ) Comparative effect of 1 μM rocaglates C9433, C5557 and C8808 on gene expression. BMDM were treated with 1 μM compound for 24 hr and the mRNA expression of Irf1, Igtp and Irgm1, Irf5, Gadd45b and Ptgs2 was measured by real-time PCR. ( e ) Effect of C9433 on IFNγ-inducible gene expression. BMDM was treated with different doses of C9433 (0.33, 1, 3.3 μM) in presence and absence of 0.2U IFNγ for 24 hrs and mRNA expression of Irf7, Irf1 and Ido1 was measured by real-time PCR. Cells treated with 20 U/mL IFNγ served as a positive control of gene expression. PCR data are representative of at least two independent experiments.

    Journal: Scientific Reports

    Article Title: Fine-tuning of macrophage activation using synthetic rocaglate derivatives

    doi: 10.1038/srep24409

    Figure Lengend Snippet: CMLD candidate compound identification and validation. ( a ) Dose response in J774-21 GFP-Ipr1 reporter cell line. J7-21 cells were treated with different doses of C9433 (0.010, 0.033, 0.1, 0.33, 1, 3.3 μM) in the presence of 0.2 U/mL IFNγ for 24 hrs, followed by addition of 1 μg/mL dox for 24 hrs. GFP-Ipr1 expression was measured using automated cytometry. Results are representative of at least two independent experiments performed in triplicates. ( b ) Toxicity of compounds in primary BMDM. BMDM were treated with compounds C9433, C5557 and C8808 at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. ( c ) Dose-dependent translation inhibition by rocaglates. 293TR-FLuc cells were treated with rocaglates C9433, C5557 and C8808 (0.033, 0.1, 0.33, 1, 3.3 μM) and luciferase activity was measured after 18 hrs. Two independent experiments were performed in triplicates. ( d ) Comparative effect of 1 μM rocaglates C9433, C5557 and C8808 on gene expression. BMDM were treated with 1 μM compound for 24 hr and the mRNA expression of Irf1, Igtp and Irgm1, Irf5, Gadd45b and Ptgs2 was measured by real-time PCR. ( e ) Effect of C9433 on IFNγ-inducible gene expression. BMDM was treated with different doses of C9433 (0.33, 1, 3.3 μM) in presence and absence of 0.2U IFNγ for 24 hrs and mRNA expression of Irf7, Irf1 and Ido1 was measured by real-time PCR. Cells treated with 20 U/mL IFNγ served as a positive control of gene expression. PCR data are representative of at least two independent experiments.

    Article Snippet: IRF1 antibody was obtained from Cell Signalling (1:500).

    Techniques: Expressing, Cytometry, Inhibition, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction

    Comparative activity and characterization of new rocaglates. ( a ) BMDM were treated with 1 μM compound (1-C9433, 2-C8809, 3-C10021, 4-C7564, 5-C7565, and 6-C10361) alone and in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irgm2, Irf5 and Ptgs2 was measured by qPCR. Data is presented relative to expression in untreated cells (set as 1). ( b ) BMDM were treated with 0.33 μM compounds (mentioned above) in presence and/or absence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR. Data is presented relative to expression in 0.2U IFNγ treated cells for Irf1 and untreated for Irf5 and Ptgs2 (set as 1). ( c ) 293TR-FLuc cells were treated with 0.33 μM compounds (mentioned above) and luciferase activity was measured after 18 hrs. Data represents values from two independent experiments. ( d ) BMDM were treated with different doses of C9433 and C8809 in presence of 0.2 U/mL IFNγ for 24 hrs. mRNA expression of Irf1 was measured by qPCR. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1). ( e ) 293 T-FLuc cells were treated with different doses of C9433 and C8809 and the luciferase activity was measured after 18 hrs in triplicates. ( f ) BMDM were treated with C8809 and C9433 for 24 hrs in the presence and absence of 0.2 U/mL IFNγ and IRF1 induction was detected by immunoblotting. 20 U/mL IFNγ treated cells served as positive controls. ( g ) BMDM were treated with different doses of C8809 and C9433 for 6 hrs and probed with anti-LC3B mAb. A mix of chloroquine(10 μM) and rapamycin(500 nM) served as positive control (C). All immunoblots are representative of at least two independent experiments. ( h ) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence of different doses of C8809 and NO production was measured in triplicates. ( i ) BMDM were treated with 10 ng/ml TNFα in the presence of C8809 for 24 hrs. mRNA expression of IFNβ, IP10 and IL10 were measured by q-PCR. Data is calculated as % of gene expression relative to 10 ng/mL TNFα treated cells. All q-PCR results were normalized to expression of 18S and are representative of at least two independent experiments.

    Journal: Scientific Reports

    Article Title: Fine-tuning of macrophage activation using synthetic rocaglate derivatives

    doi: 10.1038/srep24409

    Figure Lengend Snippet: Comparative activity and characterization of new rocaglates. ( a ) BMDM were treated with 1 μM compound (1-C9433, 2-C8809, 3-C10021, 4-C7564, 5-C7565, and 6-C10361) alone and in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irgm2, Irf5 and Ptgs2 was measured by qPCR. Data is presented relative to expression in untreated cells (set as 1). ( b ) BMDM were treated with 0.33 μM compounds (mentioned above) in presence and/or absence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR. Data is presented relative to expression in 0.2U IFNγ treated cells for Irf1 and untreated for Irf5 and Ptgs2 (set as 1). ( c ) 293TR-FLuc cells were treated with 0.33 μM compounds (mentioned above) and luciferase activity was measured after 18 hrs. Data represents values from two independent experiments. ( d ) BMDM were treated with different doses of C9433 and C8809 in presence of 0.2 U/mL IFNγ for 24 hrs. mRNA expression of Irf1 was measured by qPCR. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1). ( e ) 293 T-FLuc cells were treated with different doses of C9433 and C8809 and the luciferase activity was measured after 18 hrs in triplicates. ( f ) BMDM were treated with C8809 and C9433 for 24 hrs in the presence and absence of 0.2 U/mL IFNγ and IRF1 induction was detected by immunoblotting. 20 U/mL IFNγ treated cells served as positive controls. ( g ) BMDM were treated with different doses of C8809 and C9433 for 6 hrs and probed with anti-LC3B mAb. A mix of chloroquine(10 μM) and rapamycin(500 nM) served as positive control (C). All immunoblots are representative of at least two independent experiments. ( h ) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence of different doses of C8809 and NO production was measured in triplicates. ( i ) BMDM were treated with 10 ng/ml TNFα in the presence of C8809 for 24 hrs. mRNA expression of IFNβ, IP10 and IL10 were measured by q-PCR. Data is calculated as % of gene expression relative to 10 ng/mL TNFα treated cells. All q-PCR results were normalized to expression of 18S and are representative of at least two independent experiments.

    Article Snippet: IRF1 antibody was obtained from Cell Signalling (1:500).

    Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Positive Control, Western Blot, Polymerase Chain Reaction

    Comaparative activity of known rocaglates and translational inhibitors. ( a ) BMDM were treated with 0.33 μM compounds in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1 was measured by qPCR and normalized to expression of 18 S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments. ( b ) BMDM were treated with silvestrol and RHT at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. ( c ) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence and absence of 1 μM compounds and production of NO (assayed as NO 2 − ) was determined. All measurements for NO production were performed in triplicates. ( d ) BMDM were treated with 1 μM compounds for 6 hrs and autophagy was determined by increase of LC3B-II to LC3B-I ratio by immunoblotting. Blots represent data of two independent experiments. 293 T-FLuc cells were treated with different doses of ( e ) silvestrol, RHT, exo-RHT and ent -RHT and ( f ) cycloheximide and rapamycin. The luciferase activity was measured after 18 hrs. Data represents values from experiment performed in triplicates. ( g ) BMDM were treated with cycloheximide and rapamycin at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. ( h ) BMDM were treated with 0.33 μM compounds in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR and normalized to expression of 18 S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments. ( i ) BMDM were treated with 0.33, 1 and 3.3 μM rapamycin in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR and normalized to expression of 18S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments.

    Journal: Scientific Reports

    Article Title: Fine-tuning of macrophage activation using synthetic rocaglate derivatives

    doi: 10.1038/srep24409

    Figure Lengend Snippet: Comaparative activity of known rocaglates and translational inhibitors. ( a ) BMDM were treated with 0.33 μM compounds in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1 was measured by qPCR and normalized to expression of 18 S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments. ( b ) BMDM were treated with silvestrol and RHT at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. ( c ) BMDM were treated with 15 ng/mL TNFα and 10 U/mL IFNγ for 24 hrs in the presence and absence of 1 μM compounds and production of NO (assayed as NO 2 − ) was determined. All measurements for NO production were performed in triplicates. ( d ) BMDM were treated with 1 μM compounds for 6 hrs and autophagy was determined by increase of LC3B-II to LC3B-I ratio by immunoblotting. Blots represent data of two independent experiments. 293 T-FLuc cells were treated with different doses of ( e ) silvestrol, RHT, exo-RHT and ent -RHT and ( f ) cycloheximide and rapamycin. The luciferase activity was measured after 18 hrs. Data represents values from experiment performed in triplicates. ( g ) BMDM were treated with cycloheximide and rapamycin at concentrations shown for 24 hrs and % of PI positive cells were calculated. Data is represented as % of survival of two independent experiments performed in duplicates. ( h ) BMDM were treated with 0.33 μM compounds in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR and normalized to expression of 18 S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments. ( i ) BMDM were treated with 0.33, 1 and 3.3 μM rapamycin in presence of 0.2 U/mL IFNγ for 24 hr. mRNA expression of Irf1, Irf5 and Ptgs2 was measured by qPCR and normalized to expression of 18S. Data is presented relative to expression in 0.2U IFNγ treated cells (set as 1) and represents results from two independent experiments.

    Article Snippet: IRF1 antibody was obtained from Cell Signalling (1:500).

    Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Luciferase

    miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P

    Journal: OncoTargets and therapy

    Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

    doi: 10.2147/OTT.S238975

    Figure Lengend Snippet: miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P

    Article Snippet: Membranes were incubated with primary antibody against IRF1 (#8478, Cell Signaling Technology) or β-actin (#3700, Cell Signaling Technology) overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody.

    Techniques: Over Expression, Expressing

    miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P

    Journal: OncoTargets and therapy

    Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

    doi: 10.2147/OTT.S238975

    Figure Lengend Snippet: miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P

    Article Snippet: Membranes were incubated with primary antibody against IRF1 (#8478, Cell Signaling Technology) or β-actin (#3700, Cell Signaling Technology) overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody.

    Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Quantitative RT-PCR

    IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P

    Journal: OncoTargets and therapy

    Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

    doi: 10.2147/OTT.S238975

    Figure Lengend Snippet: IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P

    Article Snippet: Membranes were incubated with primary antibody against IRF1 (#8478, Cell Signaling Technology) or β-actin (#3700, Cell Signaling Technology) overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody.

    Techniques: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Inhibition

    IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.

    Journal: OncoTargets and therapy

    Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

    doi: 10.2147/OTT.S238975

    Figure Lengend Snippet: IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.

    Article Snippet: Membranes were incubated with primary antibody against IRF1 (#8478, Cell Signaling Technology) or β-actin (#3700, Cell Signaling Technology) overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody.

    Techniques: Expressing, Blocking Assay

    Expression of interferon regulatory factor-1 (IRF-1) is suppressed in hepatocellular carcinoma (HCC). (A) IRF-1 mRNA expression in 7 cases of HCC was decreased compared with the expression level in adjacent non-cancerous background liver samples. The IRF-1 mRNA level was quantified by qPCR. (B) IRF-1 mRNA levels were significantly lower in the HCC compared to the non-cancerous liver samples ( ** p

    Journal: Oncology Reports

    Article Title: MicroRNA-23a downregulates the expression of interferon regulatory factor-1 in hepatocellular carcinoma cells

    doi: 10.3892/or.2016.4864

    Figure Lengend Snippet: Expression of interferon regulatory factor-1 (IRF-1) is suppressed in hepatocellular carcinoma (HCC). (A) IRF-1 mRNA expression in 7 cases of HCC was decreased compared with the expression level in adjacent non-cancerous background liver samples. The IRF-1 mRNA level was quantified by qPCR. (B) IRF-1 mRNA levels were significantly lower in the HCC compared to the non-cancerous liver samples ( ** p

    Article Snippet: Huh-7 cells were cultured on coverslips, fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, permeabilized with 0.1% Triton X-100 and 10% FBS in PBS for 30 min at room temperature, and incubated with the primary IRF-1 antibodies (Cell Signaling Technology) for 1 h, which was diluted in a 1:150 ratio.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Expression of miR-23a is inversely correlated with IRF-1 mRNA in (A) primary human hepatocytes (hHC) and (B) HCC Huh-7 cells induced by IFNγ (250 IU/ml) for 3–24 h. Results shown are representative of three similar experiments.

    Journal: Oncology Reports

    Article Title: MicroRNA-23a downregulates the expression of interferon regulatory factor-1 in hepatocellular carcinoma cells

    doi: 10.3892/or.2016.4864

    Figure Lengend Snippet: Expression of miR-23a is inversely correlated with IRF-1 mRNA in (A) primary human hepatocytes (hHC) and (B) HCC Huh-7 cells induced by IFNγ (250 IU/ml) for 3–24 h. Results shown are representative of three similar experiments.

    Article Snippet: Huh-7 cells were cultured on coverslips, fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, permeabilized with 0.1% Triton X-100 and 10% FBS in PBS for 30 min at room temperature, and incubated with the primary IRF-1 antibodies (Cell Signaling Technology) for 1 h, which was diluted in a 1:150 ratio.

    Techniques: Expressing

    IRF-1 nuclear protein in Huh-7 liver tumor cells is increased by the miR-23a inhibitor. (A) Immunofluorescent staining of IRF-1 nuclear protein in Huh-7 cells shows low basal expression (scale bar, 10 µ m). (B) IFNγ (250 IU/ml, 6 h) strongly induced IRF-1 nuclear protein expression in the Huh-7 cells. (C) Expression of miR-23a inhibitor negative control (admiR-23a NC infection for 48 h) did not alter basal IRF-1 protein. (D) Expression of miR-23a inhibitor (admiR-23a inhibitor infection for 48 h) increased basal IRF-1 protein.

    Journal: Oncology Reports

    Article Title: MicroRNA-23a downregulates the expression of interferon regulatory factor-1 in hepatocellular carcinoma cells

    doi: 10.3892/or.2016.4864

    Figure Lengend Snippet: IRF-1 nuclear protein in Huh-7 liver tumor cells is increased by the miR-23a inhibitor. (A) Immunofluorescent staining of IRF-1 nuclear protein in Huh-7 cells shows low basal expression (scale bar, 10 µ m). (B) IFNγ (250 IU/ml, 6 h) strongly induced IRF-1 nuclear protein expression in the Huh-7 cells. (C) Expression of miR-23a inhibitor negative control (admiR-23a NC infection for 48 h) did not alter basal IRF-1 protein. (D) Expression of miR-23a inhibitor (admiR-23a inhibitor infection for 48 h) increased basal IRF-1 protein.

    Article Snippet: Huh-7 cells were cultured on coverslips, fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, permeabilized with 0.1% Triton X-100 and 10% FBS in PBS for 30 min at room temperature, and incubated with the primary IRF-1 antibodies (Cell Signaling Technology) for 1 h, which was diluted in a 1:150 ratio.

    Techniques: Staining, Expressing, Negative Control, Infection

    miR-23a regulates IRF-1 mRNA by binding to the IRF-1 3′-untranslated region (3′UTR). (A) Bioinformatic analysis indicated a putative miR-23a-specific binding site in the IRF-1 3′UTR. (B) IRF-1 3′UTR luciferase reporter activity was decreased by the miR-23a mimic, and increased by the miR-23a inhibitor in the HCC HCT116 cells ( * p

    Journal: Oncology Reports

    Article Title: MicroRNA-23a downregulates the expression of interferon regulatory factor-1 in hepatocellular carcinoma cells

    doi: 10.3892/or.2016.4864

    Figure Lengend Snippet: miR-23a regulates IRF-1 mRNA by binding to the IRF-1 3′-untranslated region (3′UTR). (A) Bioinformatic analysis indicated a putative miR-23a-specific binding site in the IRF-1 3′UTR. (B) IRF-1 3′UTR luciferase reporter activity was decreased by the miR-23a mimic, and increased by the miR-23a inhibitor in the HCC HCT116 cells ( * p

    Article Snippet: Huh-7 cells were cultured on coverslips, fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, permeabilized with 0.1% Triton X-100 and 10% FBS in PBS for 30 min at room temperature, and incubated with the primary IRF-1 antibodies (Cell Signaling Technology) for 1 h, which was diluted in a 1:150 ratio.

    Techniques: Binding Assay, Luciferase, Activity Assay

    IRF-1 expression is downregulated by miR-23a. IRF-1 mRNA expression as determined by real-time PCR was induced by IFNγ stimulation in (A) Huh-7 and (B) HepG2 cells. miR-23a inhibitor increased basal IRF-1 mRNA levels, while the miR-23a negative control (NC) had no effect. (C) The basal IRF-1 nuclear protein level in the HepG2 cells was increased by the miR-23a inhibitor. In contrast, the miR-23a mimic decreased the IFNγ-induced IRF-1 nuclear protein level, while the miR-23a inhibitor had no significant effect compared to IFNγ alone. IRF-1 protein levels were measured by western blotting. IFNγ (250 IU/ml) for 6 h. Results shown are representative of two similar experiments.

    Journal: Oncology Reports

    Article Title: MicroRNA-23a downregulates the expression of interferon regulatory factor-1 in hepatocellular carcinoma cells

    doi: 10.3892/or.2016.4864

    Figure Lengend Snippet: IRF-1 expression is downregulated by miR-23a. IRF-1 mRNA expression as determined by real-time PCR was induced by IFNγ stimulation in (A) Huh-7 and (B) HepG2 cells. miR-23a inhibitor increased basal IRF-1 mRNA levels, while the miR-23a negative control (NC) had no effect. (C) The basal IRF-1 nuclear protein level in the HepG2 cells was increased by the miR-23a inhibitor. In contrast, the miR-23a mimic decreased the IFNγ-induced IRF-1 nuclear protein level, while the miR-23a inhibitor had no significant effect compared to IFNγ alone. IRF-1 protein levels were measured by western blotting. IFNγ (250 IU/ml) for 6 h. Results shown are representative of two similar experiments.

    Article Snippet: Huh-7 cells were cultured on coverslips, fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, permeabilized with 0.1% Triton X-100 and 10% FBS in PBS for 30 min at room temperature, and incubated with the primary IRF-1 antibodies (Cell Signaling Technology) for 1 h, which was diluted in a 1:150 ratio.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Negative Control, Western Blot

    Effects of wortmannin on NF- κ B and IRF-3 nuclear translocation, I κ B degradation and Akt phosphorylation in H1N1-infected A549 cells. For nuclear translocation and cytosolic I κ B degradation, cells were lysed at 16 h p.i. and then protein was extracted for western blot analysis as described in Section 2 . While assessing Akt phosphorylation, cells were lysed at 6 h p.i. then cytosolic protein was extracted. Similar results were obtained in three independent experiments.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: 8-Prenylkaempferol Suppresses Influenza A Virus-Induced RANTES Production in A549 Cells via Blocking PI3K-Mediated Transcriptional Activation of NF-κB and IRF3

    doi: 10.1093/ecam/nep066

    Figure Lengend Snippet: Effects of wortmannin on NF- κ B and IRF-3 nuclear translocation, I κ B degradation and Akt phosphorylation in H1N1-infected A549 cells. For nuclear translocation and cytosolic I κ B degradation, cells were lysed at 16 h p.i. and then protein was extracted for western blot analysis as described in Section 2 . While assessing Akt phosphorylation, cells were lysed at 6 h p.i. then cytosolic protein was extracted. Similar results were obtained in three independent experiments.

    Article Snippet: After transferring, membrane was detected by antibodies against NF-κ B p65; Iκ B-α , IRF-3 (all purchased from Santa Cruz Biotechnology, CA, USA), Akt and c-Jun (both purchased from Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Translocation Assay, Infection, Western Blot

    Effects of 8-PK on NF- κ B and IRF-3 nuclear translocation, I κ B degradation and Akt phosphorylation in H1N1-infected A549 cells. For nuclear translocation and cytosolic I κ B degradation, cells were lysed at 16 h p.i. and then protein was extracted by western blot technique as described in Section 2 . While assessing for Akt phosphorylation, cells were lysed at 6 h p.i. then cytosolic protein was extracted. Similar results were obtained in four independent experiments.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: 8-Prenylkaempferol Suppresses Influenza A Virus-Induced RANTES Production in A549 Cells via Blocking PI3K-Mediated Transcriptional Activation of NF-κB and IRF3

    doi: 10.1093/ecam/nep066

    Figure Lengend Snippet: Effects of 8-PK on NF- κ B and IRF-3 nuclear translocation, I κ B degradation and Akt phosphorylation in H1N1-infected A549 cells. For nuclear translocation and cytosolic I κ B degradation, cells were lysed at 16 h p.i. and then protein was extracted by western blot technique as described in Section 2 . While assessing for Akt phosphorylation, cells were lysed at 6 h p.i. then cytosolic protein was extracted. Similar results were obtained in four independent experiments.

    Article Snippet: After transferring, membrane was detected by antibodies against NF-κ B p65; Iκ B-α , IRF-3 (all purchased from Santa Cruz Biotechnology, CA, USA), Akt and c-Jun (both purchased from Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Translocation Assay, Infection, Western Blot

    Time-related effect of H1N1 inoculation on (a) transcriptional factors activation measured by NF- κ B and IRF-3 nuclear translocation, I κ B degradation and c-Jun phosphorylation, as well as (b) Akt phosphorylation in A549 alveolar epithelial cells, respectively. Nuclear (for NF- κ B and IRF-3) and cytosolic proteins (for I κ B and c-Jun) were extracted in the absence of virus (indicated as 0) or obtained at 1, 6, or 16 h p.i as described in Section 2 then assessed by western blotting. Similar results were obtained in four independent experiments.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: 8-Prenylkaempferol Suppresses Influenza A Virus-Induced RANTES Production in A549 Cells via Blocking PI3K-Mediated Transcriptional Activation of NF-κB and IRF3

    doi: 10.1093/ecam/nep066

    Figure Lengend Snippet: Time-related effect of H1N1 inoculation on (a) transcriptional factors activation measured by NF- κ B and IRF-3 nuclear translocation, I κ B degradation and c-Jun phosphorylation, as well as (b) Akt phosphorylation in A549 alveolar epithelial cells, respectively. Nuclear (for NF- κ B and IRF-3) and cytosolic proteins (for I κ B and c-Jun) were extracted in the absence of virus (indicated as 0) or obtained at 1, 6, or 16 h p.i as described in Section 2 then assessed by western blotting. Similar results were obtained in four independent experiments.

    Article Snippet: After transferring, membrane was detected by antibodies against NF-κ B p65; Iκ B-α , IRF-3 (all purchased from Santa Cruz Biotechnology, CA, USA), Akt and c-Jun (both purchased from Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Activation Assay, Translocation Assay, Western Blot

    Schematic diagram illustrating the signaling pathways involved in 8-PK's inhibition of RANTES production in H1N1-infected A549 cells. After viral ssRNA binding to TLR, the adaptor protein MyD88 couples TLR and IRAK. Subsequently, MyD88 mediates a signaling cascade in TLR-triggered cells that leads to activation of IKK-I κ B-NF κ B pathways and control inflammatory genes expression include RANTES. The core IKK complex consists of the kinases IKK α and IKK β and the regulatory IKK γ /NEMO protein. The activation of IKK α / β depends on phosphorylation of serines at their activation loop. This process probably involves phosphorylation by recruit upstream kinases such as TAK1 leading to the rapid phosphorylation, ubiquitination, and ultimately proteolytic degradation of I κ B, which frees NF- κ B to translocate to the nucleus, where it regulates RANTES gene transcription. The suppression of H1N1-induced RANTES production by 8-PK may occur through downregulate I κ B degradation and NF- κ B nuclear translocation. Whether 8-PK interfere undefined upstream TAK1-IKK events to suppress RANTES production needed further study. An alternative pathway regulating RANTES production is via PI3K pathway. This leads to the activation of Akt, a cytosolic serine/threonine kinase that acts downstream of PI3K, and subsequently phosphorylation of IRF-3, a transcription factor that regulate RANTES gene expression. We found that 8-PK also can target at PI3K to block PI3K-mediated Akt phosphorylation and downregulate subsequently either NF- κ B nuclear translocation or IRF-3 phosphorylation in A549 cells reciprocally to suppress RANTES production. ssRNA, single strand RNA; TLR, Toll-like receptor; MyD88, myeloid differentiation (MyD) marker; IRAK, IL-1R-activated kinase; TRAF6, TNF α receptor-associated factor 6; TAK1, transforming growth factor- β (TGF- β )-activated kinase 1; RANTES, regulated on activation, normal T cell expressed and secreted.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: 8-Prenylkaempferol Suppresses Influenza A Virus-Induced RANTES Production in A549 Cells via Blocking PI3K-Mediated Transcriptional Activation of NF-κB and IRF3

    doi: 10.1093/ecam/nep066

    Figure Lengend Snippet: Schematic diagram illustrating the signaling pathways involved in 8-PK's inhibition of RANTES production in H1N1-infected A549 cells. After viral ssRNA binding to TLR, the adaptor protein MyD88 couples TLR and IRAK. Subsequently, MyD88 mediates a signaling cascade in TLR-triggered cells that leads to activation of IKK-I κ B-NF κ B pathways and control inflammatory genes expression include RANTES. The core IKK complex consists of the kinases IKK α and IKK β and the regulatory IKK γ /NEMO protein. The activation of IKK α / β depends on phosphorylation of serines at their activation loop. This process probably involves phosphorylation by recruit upstream kinases such as TAK1 leading to the rapid phosphorylation, ubiquitination, and ultimately proteolytic degradation of I κ B, which frees NF- κ B to translocate to the nucleus, where it regulates RANTES gene transcription. The suppression of H1N1-induced RANTES production by 8-PK may occur through downregulate I κ B degradation and NF- κ B nuclear translocation. Whether 8-PK interfere undefined upstream TAK1-IKK events to suppress RANTES production needed further study. An alternative pathway regulating RANTES production is via PI3K pathway. This leads to the activation of Akt, a cytosolic serine/threonine kinase that acts downstream of PI3K, and subsequently phosphorylation of IRF-3, a transcription factor that regulate RANTES gene expression. We found that 8-PK also can target at PI3K to block PI3K-mediated Akt phosphorylation and downregulate subsequently either NF- κ B nuclear translocation or IRF-3 phosphorylation in A549 cells reciprocally to suppress RANTES production. ssRNA, single strand RNA; TLR, Toll-like receptor; MyD88, myeloid differentiation (MyD) marker; IRAK, IL-1R-activated kinase; TRAF6, TNF α receptor-associated factor 6; TAK1, transforming growth factor- β (TGF- β )-activated kinase 1; RANTES, regulated on activation, normal T cell expressed and secreted.

    Article Snippet: After transferring, membrane was detected by antibodies against NF-κ B p65; Iκ B-α , IRF-3 (all purchased from Santa Cruz Biotechnology, CA, USA), Akt and c-Jun (both purchased from Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Inhibition, Infection, Binding Assay, Activation Assay, Expressing, Translocation Assay, Blocking Assay, Marker

    (a) Inhibitory effects of pyrrolidinedithio carbamate (PDTC, a NF- κ B inhibitor), IRF-3 siRNA and wortmannin (wort, a PI3K inhibitor) on the nuclear translocation of NF- κ B/IRF-3 and phosphorylation of Akt in H1N1-infected A549 cells and (b) the same treatment on RANTES accumulation (100 nM PDTC, 100 nM siRNA and 30 μ M wortmannin). For nuclear translocation assay, cells were lysed at 16 h p.i. and nuclear protein was extracted for western blot analysis as described in Section 2 . While assessing Akt phosphorylation, cells were lysed at 6 h p.i. then cytosolic protein was extracted. For RANTES measurement, culture medium was collected at 48 h (p.i.). Data reported are mean ± SE of six independent experiments, each performed in triplicate. ** P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: 8-Prenylkaempferol Suppresses Influenza A Virus-Induced RANTES Production in A549 Cells via Blocking PI3K-Mediated Transcriptional Activation of NF-κB and IRF3

    doi: 10.1093/ecam/nep066

    Figure Lengend Snippet: (a) Inhibitory effects of pyrrolidinedithio carbamate (PDTC, a NF- κ B inhibitor), IRF-3 siRNA and wortmannin (wort, a PI3K inhibitor) on the nuclear translocation of NF- κ B/IRF-3 and phosphorylation of Akt in H1N1-infected A549 cells and (b) the same treatment on RANTES accumulation (100 nM PDTC, 100 nM siRNA and 30 μ M wortmannin). For nuclear translocation assay, cells were lysed at 16 h p.i. and nuclear protein was extracted for western blot analysis as described in Section 2 . While assessing Akt phosphorylation, cells were lysed at 6 h p.i. then cytosolic protein was extracted. For RANTES measurement, culture medium was collected at 48 h (p.i.). Data reported are mean ± SE of six independent experiments, each performed in triplicate. ** P

    Article Snippet: After transferring, membrane was detected by antibodies against NF-κ B p65; Iκ B-α , IRF-3 (all purchased from Santa Cruz Biotechnology, CA, USA), Akt and c-Jun (both purchased from Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Translocation Assay, Infection, Nuclear Translocation Assay, Western Blot

    Western blot analysis of NCoA3 and IRF8 proteins expression . Nuclear extract (100 μg) from U1, ACH-2, J1.1 and OM10.1 treated (+) or not (-) with NaB for 24 h were resolved by SDS-PAGE and immunoblotted with anti-NCoA3 or anti-IRF8 antibody, as indicated. The amount of protein was normalized using anti-actin antibody. Figures below NCoA3 immunoblot indicated the results of the quantification using Image Tool (Syngene) software of the ratio NCoA3/actin upon NaB-treatment (+) versus NCoA3/actin non-treated (-). Results are representative of three independent experiments.

    Journal: Retrovirology

    Article Title: Characterization of two candidate genes, NCoA3 and IRF8, potentially involved in the control of HIV-1 latency

    doi: 10.1186/1742-4690-2-73

    Figure Lengend Snippet: Western blot analysis of NCoA3 and IRF8 proteins expression . Nuclear extract (100 μg) from U1, ACH-2, J1.1 and OM10.1 treated (+) or not (-) with NaB for 24 h were resolved by SDS-PAGE and immunoblotted with anti-NCoA3 or anti-IRF8 antibody, as indicated. The amount of protein was normalized using anti-actin antibody. Figures below NCoA3 immunoblot indicated the results of the quantification using Image Tool (Syngene) software of the ratio NCoA3/actin upon NaB-treatment (+) versus NCoA3/actin non-treated (-). Results are representative of three independent experiments.

    Article Snippet: The expression of IRF8 inhibited by 43.5 ± 10.6 to 74.7 ± 2.5 % the IRF1-mediated activation of the ISRE-TK in a dose dependent fashion (Figure ).

    Techniques: Western Blot, Expressing, SDS Page, Software

    IRF8 represses the IRF1-mediated activation of the HIV-1 ISRE element . HEK293 cells were cotransfected with pISRE-TK-luc (250 ng, solid bars) or pISREmut-TK-luc (250 ng, white bars) with (+) or without (-) pIRF1 (250 ng), pIRF8 (1–2.5 μg), or pIRF8-DBD (1 μg) expression vectors. NLI (normalized luciferase index) were measured after 24 h and the activation folds compared to the basal activity of the pISRE-TK-luc or pISREmut-TK-luc were determined. Results represent the means of five independent experiments.

    Journal: Retrovirology

    Article Title: Characterization of two candidate genes, NCoA3 and IRF8, potentially involved in the control of HIV-1 latency

    doi: 10.1186/1742-4690-2-73

    Figure Lengend Snippet: IRF8 represses the IRF1-mediated activation of the HIV-1 ISRE element . HEK293 cells were cotransfected with pISRE-TK-luc (250 ng, solid bars) or pISREmut-TK-luc (250 ng, white bars) with (+) or without (-) pIRF1 (250 ng), pIRF8 (1–2.5 μg), or pIRF8-DBD (1 μg) expression vectors. NLI (normalized luciferase index) were measured after 24 h and the activation folds compared to the basal activity of the pISRE-TK-luc or pISREmut-TK-luc were determined. Results represent the means of five independent experiments.

    Article Snippet: The expression of IRF8 inhibited by 43.5 ± 10.6 to 74.7 ± 2.5 % the IRF1-mediated activation of the ISRE-TK in a dose dependent fashion (Figure ).

    Techniques: Activation Assay, Expressing, Luciferase, Activity Assay

    Real-time RT-PCR analysis of NCoA3 and IRF8 mRNAs expression in NaB-treated U1 and ACH-2 cells . Total RNAs were isolated from U1 or ACH-2 cells treated or not with NaB for 24 h and 48 h and real-time PCR were performed on cDNAs using gene specific primers for NCoA3 , IRF8 or Cyclophilin A . NCoA3 and IRF8 expressions were normalized to the expression of Cyclophilin A . The NCoA3 increase fold (A) in U1 (solid bars) or ACH-2 (white bars) cells and the IRF8 decrease fold (B) in U1 cells treated with NaB for 24 h and 48 h compared to non-treated (NT) cells were determined. Results represent the means of five independent experiments performed in duplicate.

    Journal: Retrovirology

    Article Title: Characterization of two candidate genes, NCoA3 and IRF8, potentially involved in the control of HIV-1 latency

    doi: 10.1186/1742-4690-2-73

    Figure Lengend Snippet: Real-time RT-PCR analysis of NCoA3 and IRF8 mRNAs expression in NaB-treated U1 and ACH-2 cells . Total RNAs were isolated from U1 or ACH-2 cells treated or not with NaB for 24 h and 48 h and real-time PCR were performed on cDNAs using gene specific primers for NCoA3 , IRF8 or Cyclophilin A . NCoA3 and IRF8 expressions were normalized to the expression of Cyclophilin A . The NCoA3 increase fold (A) in U1 (solid bars) or ACH-2 (white bars) cells and the IRF8 decrease fold (B) in U1 cells treated with NaB for 24 h and 48 h compared to non-treated (NT) cells were determined. Results represent the means of five independent experiments performed in duplicate.

    Article Snippet: The expression of IRF8 inhibited by 43.5 ± 10.6 to 74.7 ± 2.5 % the IRF1-mediated activation of the ISRE-TK in a dose dependent fashion (Figure ).

    Techniques: Quantitative RT-PCR, Expressing, Isolation, Real-time Polymerase Chain Reaction

    Real-time RT-PCR analysis of NCoA3 and IRF8 mRNAs expression in NaB-treated U937 and CEM cells . Total RNAs were isolated from U937 or CEM cells treated or not with NaB for 24 h and 48 h and real-time PCR were performed on cDNAs using gene specific primers for NCoA3 , IRF8 or Cyclophilin A . NCoA3 and IRF8 expressions were normalized to the expression of Cyclophilin A . The NCoA3 increase fold (A) in U937 (solid bars) or CEM (white bars) cells and the IRF8 decrease fold (B) in U937 cells treated with NaB for 24 h and 48 h compared to non-treated (NT) cells were determined. Results represent the means of five independent experiments performed in duplicate.

    Journal: Retrovirology

    Article Title: Characterization of two candidate genes, NCoA3 and IRF8, potentially involved in the control of HIV-1 latency

    doi: 10.1186/1742-4690-2-73

    Figure Lengend Snippet: Real-time RT-PCR analysis of NCoA3 and IRF8 mRNAs expression in NaB-treated U937 and CEM cells . Total RNAs were isolated from U937 or CEM cells treated or not with NaB for 24 h and 48 h and real-time PCR were performed on cDNAs using gene specific primers for NCoA3 , IRF8 or Cyclophilin A . NCoA3 and IRF8 expressions were normalized to the expression of Cyclophilin A . The NCoA3 increase fold (A) in U937 (solid bars) or CEM (white bars) cells and the IRF8 decrease fold (B) in U937 cells treated with NaB for 24 h and 48 h compared to non-treated (NT) cells were determined. Results represent the means of five independent experiments performed in duplicate.

    Article Snippet: The expression of IRF8 inhibited by 43.5 ± 10.6 to 74.7 ± 2.5 % the IRF1-mediated activation of the ISRE-TK in a dose dependent fashion (Figure ).

    Techniques: Quantitative RT-PCR, Expressing, Isolation, Real-time Polymerase Chain Reaction

    Real-time RT-PCR analysis of NCoA3 and IRF8 mRNAs expression in PMA- or TSA-treated U1 and ACH-2 cells . Total RNAs were isolated from U1 or ACH-2 cells treated or not with PMA for 24 h and 48 h or TSA for 24 h and real-time PCR were performed on cDNAs using gene specific primers for NCoA3 , IRF8 or Cyclophilin A . NCoA3 and IRF8 expressions were normalized to the expression of Cyclophilin A . The NCoA3 increase fold (A) in U1 (solid bars) or ACH-2 (white bars) cells treated with PMA for 24 h and 48 h and the IRF8 decrease fold (B) in U1 cells treated with PMA or TSA for 24 h compared to non-treated (NT) cells were determined. Results represent the means of three independent experiments performed in duplicate.

    Journal: Retrovirology

    Article Title: Characterization of two candidate genes, NCoA3 and IRF8, potentially involved in the control of HIV-1 latency

    doi: 10.1186/1742-4690-2-73

    Figure Lengend Snippet: Real-time RT-PCR analysis of NCoA3 and IRF8 mRNAs expression in PMA- or TSA-treated U1 and ACH-2 cells . Total RNAs were isolated from U1 or ACH-2 cells treated or not with PMA for 24 h and 48 h or TSA for 24 h and real-time PCR were performed on cDNAs using gene specific primers for NCoA3 , IRF8 or Cyclophilin A . NCoA3 and IRF8 expressions were normalized to the expression of Cyclophilin A . The NCoA3 increase fold (A) in U1 (solid bars) or ACH-2 (white bars) cells treated with PMA for 24 h and 48 h and the IRF8 decrease fold (B) in U1 cells treated with PMA or TSA for 24 h compared to non-treated (NT) cells were determined. Results represent the means of three independent experiments performed in duplicate.

    Article Snippet: The expression of IRF8 inhibited by 43.5 ± 10.6 to 74.7 ± 2.5 % the IRF1-mediated activation of the ISRE-TK in a dose dependent fashion (Figure ).

    Techniques: Quantitative RT-PCR, Expressing, Isolation, Real-time Polymerase Chain Reaction

    Analysis of HIV gag , NCoA3 , and IRF8 mRNA expression after NaB stimulation on U1 and ACH-2 cells . U1 (A and B) and ACH-2 (C) cells were stimulated with 10 mM NaB and 5.10 6 cells were taken at t = 0, 4, 8, 16, 24, 48 h for RNA extraction to perform qRT-PCR. NCoA3 (A and C), IRF8 (B) and gag (A, B and C) mRNA contents were measured. Cylophilin A was used as internal standard. Results represent a representative experiment performed in duplicate.

    Journal: Retrovirology

    Article Title: Characterization of two candidate genes, NCoA3 and IRF8, potentially involved in the control of HIV-1 latency

    doi: 10.1186/1742-4690-2-73

    Figure Lengend Snippet: Analysis of HIV gag , NCoA3 , and IRF8 mRNA expression after NaB stimulation on U1 and ACH-2 cells . U1 (A and B) and ACH-2 (C) cells were stimulated with 10 mM NaB and 5.10 6 cells were taken at t = 0, 4, 8, 16, 24, 48 h for RNA extraction to perform qRT-PCR. NCoA3 (A and C), IRF8 (B) and gag (A, B and C) mRNA contents were measured. Cylophilin A was used as internal standard. Results represent a representative experiment performed in duplicate.

    Article Snippet: The expression of IRF8 inhibited by 43.5 ± 10.6 to 74.7 ± 2.5 % the IRF1-mediated activation of the ISRE-TK in a dose dependent fashion (Figure ).

    Techniques: Expressing, RNA Extraction, Quantitative RT-PCR

    NLRX1 acts independently of ROS response to promote H. capsulatum -induced LC3 conversion in macrophages. (A,B,F) Macrophages from WT and Nlrx1 −/− mice were stimulated with or without (0 min) zymosan (50 μg/ml) (A) or H. capsulatum (MOI = 5) (B,F) for indicated time. Cell lysates were extracted and analyzed by Western blotting for the expression of indicated proteins. Data shown in the lower panel are relative intensity of LC3-II normalized against the corresponding β-actin, mean ± SEM are shown ( n = 3) (A,B) . (C,D) Macrophages from WT and Nlrx1 −/− mice were stimulated with H. capsulatum for 1 h. Cell were fixed and stained for LC3B (green), F-actin (red), and nucleus compartment (blue), and viewed under confocal microscope (C) . Asterisks in the DIC/Nucleus field point to H. capsulatum yeasts. The intensity of different fluorochromes along the white arrow in the merged image is shown as the histogram on the right. The mean fluorescence intensity (MFI) of LC3 in cells engulfing H. capsulatum was quantified. Phagosomes in each cell were counted and the percentages of LC3 + phagosome are shown as mean ± SEM of 3 independent experiments (D) . (E) Macrophages from WT and Nlrx1 −/− mice were allowed to phagocytose FITC-labeled H. capsulatum (MOI = 10) for 1 h. Percentages of cells engulfing H. capsulatum were analyzed by flow cytometry ( n = 7). (G) Macrophages from WT and Nlrx1 −/− mice were preloaded with CM-H 2 DCFDA for 30 min prior to stimulation with H. capsulatum (MOI = 10) for 20 min. Flow cytometry was performed to assess global ROS production. Data shown are the percentages of ROS + cells (left panel) and the mean fluorescence intensity (MFI) (right panel) of total live cells ( n = 4). Bars represent the mean ± SEM. ** p ≤ 0.01, *** p ≤ 0.001 [ANOVA with Bonferroni's multiple comparisons post-hoc test (A,B, G) ; 2-tailed t -test (D,E) ].

    Journal: Frontiers in Immunology

    Article Title: NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages

    doi: 10.3389/fimmu.2018.02761

    Figure Lengend Snippet: NLRX1 acts independently of ROS response to promote H. capsulatum -induced LC3 conversion in macrophages. (A,B,F) Macrophages from WT and Nlrx1 −/− mice were stimulated with or without (0 min) zymosan (50 μg/ml) (A) or H. capsulatum (MOI = 5) (B,F) for indicated time. Cell lysates were extracted and analyzed by Western blotting for the expression of indicated proteins. Data shown in the lower panel are relative intensity of LC3-II normalized against the corresponding β-actin, mean ± SEM are shown ( n = 3) (A,B) . (C,D) Macrophages from WT and Nlrx1 −/− mice were stimulated with H. capsulatum for 1 h. Cell were fixed and stained for LC3B (green), F-actin (red), and nucleus compartment (blue), and viewed under confocal microscope (C) . Asterisks in the DIC/Nucleus field point to H. capsulatum yeasts. The intensity of different fluorochromes along the white arrow in the merged image is shown as the histogram on the right. The mean fluorescence intensity (MFI) of LC3 in cells engulfing H. capsulatum was quantified. Phagosomes in each cell were counted and the percentages of LC3 + phagosome are shown as mean ± SEM of 3 independent experiments (D) . (E) Macrophages from WT and Nlrx1 −/− mice were allowed to phagocytose FITC-labeled H. capsulatum (MOI = 10) for 1 h. Percentages of cells engulfing H. capsulatum were analyzed by flow cytometry ( n = 7). (G) Macrophages from WT and Nlrx1 −/− mice were preloaded with CM-H 2 DCFDA for 30 min prior to stimulation with H. capsulatum (MOI = 10) for 20 min. Flow cytometry was performed to assess global ROS production. Data shown are the percentages of ROS + cells (left panel) and the mean fluorescence intensity (MFI) (right panel) of total live cells ( n = 4). Bars represent the mean ± SEM. ** p ≤ 0.01, *** p ≤ 0.001 [ANOVA with Bonferroni's multiple comparisons post-hoc test (A,B, G) ; 2-tailed t -test (D,E) ].

    Article Snippet: Antibodies against LC3, Rubicon, NLRX1, phospho (p)-p40-phox (Thr154), p-JNK (Thr183/Tyr185), p-ERK1/2 (Thr202/Tyr204), p-p38 (Thr180/Tyr182), p-c-Fos (Ser32), p-IKKα/β (Ser176/180), p-IκBα (Ser32), IκBα, and p-NFκBp65 (Ser536) were purchased from Cell Signaling.

    Techniques: Mouse Assay, Western Blot, Expressing, Staining, Microscopy, Fluorescence, Labeling, Flow Cytometry, Cytometry

    NLRX1-TUFM is required for activation of MAPKs-AP-1 pathway and proinflammatory cytokine response to H. capsulatum . (A–C) Macrophages from WT and Nlrx1 −/− mice were stimulated with or without (0 min or medium) H. capsulatum (MOI = 5). Cell lysates were collected at 15, 30, 60, and 120 min after stimulation and subjected to Western blotting for the analysis of the indicated proteins (A) . Relative intensity of indicated protein normalized against the corresponding β-actin was shown in (B) ( n = 3). Supernatants were harvested at 18 h after stimulation, and the concentrations of TNF and IL-6 in the supernatants were quantified by ELISA ( n = 11) (C) . (D,E) Macrophages from WT mice were transfected with control siRNA or siRNA against TUFM (50 nM) for 72 h. Cells were then stimulated with or without (0 min) H. capsulatum (MOI = 5). Cell lysates were collected at 30 and 60 min after stimulation and assessed by Western blotting for the analysis of the indicated proteins (D) . Supernatants were harvested at 18 h after stimulation, and the concentrations of TNF and IL-6 in the supernatants were quantified by ELISA and are presented as the relative levels of TNF and IL-6 ( n = 6) (E) . Bars represent the mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 [2-tailed t -test (B,E) ; ANOVA with Bonferroni's multiple comparisons post-hoc test (C) ].

    Journal: Frontiers in Immunology

    Article Title: NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages

    doi: 10.3389/fimmu.2018.02761

    Figure Lengend Snippet: NLRX1-TUFM is required for activation of MAPKs-AP-1 pathway and proinflammatory cytokine response to H. capsulatum . (A–C) Macrophages from WT and Nlrx1 −/− mice were stimulated with or without (0 min or medium) H. capsulatum (MOI = 5). Cell lysates were collected at 15, 30, 60, and 120 min after stimulation and subjected to Western blotting for the analysis of the indicated proteins (A) . Relative intensity of indicated protein normalized against the corresponding β-actin was shown in (B) ( n = 3). Supernatants were harvested at 18 h after stimulation, and the concentrations of TNF and IL-6 in the supernatants were quantified by ELISA ( n = 11) (C) . (D,E) Macrophages from WT mice were transfected with control siRNA or siRNA against TUFM (50 nM) for 72 h. Cells were then stimulated with or without (0 min) H. capsulatum (MOI = 5). Cell lysates were collected at 30 and 60 min after stimulation and assessed by Western blotting for the analysis of the indicated proteins (D) . Supernatants were harvested at 18 h after stimulation, and the concentrations of TNF and IL-6 in the supernatants were quantified by ELISA and are presented as the relative levels of TNF and IL-6 ( n = 6) (E) . Bars represent the mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 [2-tailed t -test (B,E) ; ANOVA with Bonferroni's multiple comparisons post-hoc test (C) ].

    Article Snippet: Antibodies against LC3, Rubicon, NLRX1, phospho (p)-p40-phox (Thr154), p-JNK (Thr183/Tyr185), p-ERK1/2 (Thr202/Tyr204), p-p38 (Thr180/Tyr182), p-c-Fos (Ser32), p-IKKα/β (Ser176/180), p-IκBα (Ser32), IκBα, and p-NFκBp65 (Ser536) were purchased from Cell Signaling.

    Techniques: Activation Assay, Mouse Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection

    NLRX1 promotes LAP through association with TUFM-ATG5-ATG12 complex. (A) Macrophages from WT mice were stimulated with or without H. capsulatum . Cell lysates were collected at 30 min after stimulation and used for immunoprecipitation with anti-NLRX1, anti-TUFM or isotype control antibodies, followed by immunoblotting with indicated antibodies. IP, IB, and WCL denote immunoprecipitation, immunoblotting, and whole-cell lysate, respectively. (B) Macrophages were stimulated with or without H. capsulatum (MOI = 5) for 60 min. Cells were fixed and stained for NLRX1 (red), TUFM (green), F-actin (violet), and nucleus compartment (blue). Cells were viewed under confocal microscope. Asterisks in the DIC/Nucleus field point to H. capsulatum yeasts. Box areas are shown at higher magnification in the bottom left corner of the corresponding image. The intensity of different fluorochromes along the white arrow in the merged image is shown as histogram on the right. (C) Macrophages from WT mice were transfected with control siRNA or siRNA against TUFM (50 nM) for 72 h. Cells were then stimulated with or without (0 min) H. capsulatum (MOI = 5) for 30 and 60 min. After stimulation, cell lysates were collected and assessed by Western blotting. (D) Macrophages from WT and Nlrx1 −/− mice were treated with or without DPI (5 μM) for 1 h prior to stimulation with H. capsulatum (MOI = 5). Cell lysates were collected after 1 h of stimulation and analyzed by Western blotting. Data shown in the lower panel of (C,D) are relative intensity of LC3-II normalized against the corresponding β-actin, mean ± SEM are shown ( n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 [ANOVA with Bonferroni's multiple comparisons post-hoc test (C,D) ].

    Journal: Frontiers in Immunology

    Article Title: NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages

    doi: 10.3389/fimmu.2018.02761

    Figure Lengend Snippet: NLRX1 promotes LAP through association with TUFM-ATG5-ATG12 complex. (A) Macrophages from WT mice were stimulated with or without H. capsulatum . Cell lysates were collected at 30 min after stimulation and used for immunoprecipitation with anti-NLRX1, anti-TUFM or isotype control antibodies, followed by immunoblotting with indicated antibodies. IP, IB, and WCL denote immunoprecipitation, immunoblotting, and whole-cell lysate, respectively. (B) Macrophages were stimulated with or without H. capsulatum (MOI = 5) for 60 min. Cells were fixed and stained for NLRX1 (red), TUFM (green), F-actin (violet), and nucleus compartment (blue). Cells were viewed under confocal microscope. Asterisks in the DIC/Nucleus field point to H. capsulatum yeasts. Box areas are shown at higher magnification in the bottom left corner of the corresponding image. The intensity of different fluorochromes along the white arrow in the merged image is shown as histogram on the right. (C) Macrophages from WT mice were transfected with control siRNA or siRNA against TUFM (50 nM) for 72 h. Cells were then stimulated with or without (0 min) H. capsulatum (MOI = 5) for 30 and 60 min. After stimulation, cell lysates were collected and assessed by Western blotting. (D) Macrophages from WT and Nlrx1 −/− mice were treated with or without DPI (5 μM) for 1 h prior to stimulation with H. capsulatum (MOI = 5). Cell lysates were collected after 1 h of stimulation and analyzed by Western blotting. Data shown in the lower panel of (C,D) are relative intensity of LC3-II normalized against the corresponding β-actin, mean ± SEM are shown ( n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 [ANOVA with Bonferroni's multiple comparisons post-hoc test (C,D) ].

    Article Snippet: Antibodies against LC3, Rubicon, NLRX1, phospho (p)-p40-phox (Thr154), p-JNK (Thr183/Tyr185), p-ERK1/2 (Thr202/Tyr204), p-p38 (Thr180/Tyr182), p-c-Fos (Ser32), p-IKKα/β (Ser176/180), p-IκBα (Ser32), IκBα, and p-NFκBp65 (Ser536) were purchased from Cell Signaling.

    Techniques: Mouse Assay, Immunoprecipitation, Staining, Microscopy, Transfection, Western Blot

    Schematic model of LAP in H. capsulatum -infected macrophage. Upon macrophage encountering H. capsulatum , recognition of fungal β-glucan by Dectin-1 induces Syk phosphorylation. Activation of Syk triggers ROS production through NADPH oxidase. ROS is required for converting LC3-I to LC3-II on phagosomal membrane. Through a mechanism independent of ROS, NLRX1 promotes fungus-induced LAP through its association with TUFM that interacts with ATG5-ATG12 conjugate for LAPosome formation. Both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. The formation of LAPosome promotes downstream MAPKs and AP-1 activation, leading to production of anti-fungal proinflammatory cytokines.

    Journal: Frontiers in Immunology

    Article Title: NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages

    doi: 10.3389/fimmu.2018.02761

    Figure Lengend Snippet: Schematic model of LAP in H. capsulatum -infected macrophage. Upon macrophage encountering H. capsulatum , recognition of fungal β-glucan by Dectin-1 induces Syk phosphorylation. Activation of Syk triggers ROS production through NADPH oxidase. ROS is required for converting LC3-I to LC3-II on phagosomal membrane. Through a mechanism independent of ROS, NLRX1 promotes fungus-induced LAP through its association with TUFM that interacts with ATG5-ATG12 conjugate for LAPosome formation. Both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. The formation of LAPosome promotes downstream MAPKs and AP-1 activation, leading to production of anti-fungal proinflammatory cytokines.

    Article Snippet: Antibodies against LC3, Rubicon, NLRX1, phospho (p)-p40-phox (Thr154), p-JNK (Thr183/Tyr185), p-ERK1/2 (Thr202/Tyr204), p-p38 (Thr180/Tyr182), p-c-Fos (Ser32), p-IKKα/β (Ser176/180), p-IκBα (Ser32), IκBα, and p-NFκBp65 (Ser536) were purchased from Cell Signaling.

    Techniques: Infection, Activation Assay

    NLRX1 acts independently of ROS response to promote H. capsulatum -induced LC3 conversion in macrophages. (A,B,F) Macrophages from WT and Nlrx1 −/− mice were stimulated with or without (0 min) zymosan (50 μg/ml) (A) or H. capsulatum (MOI = 5) (B,F) for indicated time. Cell lysates were extracted and analyzed by Western blotting for the expression of indicated proteins. Data shown in the lower panel are relative intensity of LC3-II normalized against the corresponding β-actin, mean ± SEM are shown ( n = 3) (A,B) . (C,D) Macrophages from WT and Nlrx1 −/− mice were stimulated with H. capsulatum for 1 h. Cell were fixed and stained for LC3B (green), F-actin (red), and nucleus compartment (blue), and viewed under confocal microscope (C) . Asterisks in the DIC/Nucleus field point to H. capsulatum yeasts. The intensity of different fluorochromes along the white arrow in the merged image is shown as the histogram on the right. The mean fluorescence intensity (MFI) of LC3 in cells engulfing H. capsulatum was quantified. Phagosomes in each cell were counted and the percentages of LC3 + phagosome are shown as mean ± SEM of 3 independent experiments (D) . (E) Macrophages from WT and Nlrx1 −/− mice were allowed to phagocytose FITC-labeled H. capsulatum (MOI = 10) for 1 h. Percentages of cells engulfing H. capsulatum were analyzed by flow cytometry ( n = 7). (G) Macrophages from WT and Nlrx1 −/− mice were preloaded with CM-H 2 DCFDA for 30 min prior to stimulation with H. capsulatum (MOI = 10) for 20 min. Flow cytometry was performed to assess global ROS production. Data shown are the percentages of ROS + cells (left panel) and the mean fluorescence intensity (MFI) (right panel) of total live cells ( n = 4). Bars represent the mean ± SEM. ** p ≤ 0.01, *** p ≤ 0.001 [ANOVA with Bonferroni's multiple comparisons post-hoc test (A,B, G) ; 2-tailed t -test (D,E) ].

    Journal: Frontiers in Immunology

    Article Title: NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages

    doi: 10.3389/fimmu.2018.02761

    Figure Lengend Snippet: NLRX1 acts independently of ROS response to promote H. capsulatum -induced LC3 conversion in macrophages. (A,B,F) Macrophages from WT and Nlrx1 −/− mice were stimulated with or without (0 min) zymosan (50 μg/ml) (A) or H. capsulatum (MOI = 5) (B,F) for indicated time. Cell lysates were extracted and analyzed by Western blotting for the expression of indicated proteins. Data shown in the lower panel are relative intensity of LC3-II normalized against the corresponding β-actin, mean ± SEM are shown ( n = 3) (A,B) . (C,D) Macrophages from WT and Nlrx1 −/− mice were stimulated with H. capsulatum for 1 h. Cell were fixed and stained for LC3B (green), F-actin (red), and nucleus compartment (blue), and viewed under confocal microscope (C) . Asterisks in the DIC/Nucleus field point to H. capsulatum yeasts. The intensity of different fluorochromes along the white arrow in the merged image is shown as the histogram on the right. The mean fluorescence intensity (MFI) of LC3 in cells engulfing H. capsulatum was quantified. Phagosomes in each cell were counted and the percentages of LC3 + phagosome are shown as mean ± SEM of 3 independent experiments (D) . (E) Macrophages from WT and Nlrx1 −/− mice were allowed to phagocytose FITC-labeled H. capsulatum (MOI = 10) for 1 h. Percentages of cells engulfing H. capsulatum were analyzed by flow cytometry ( n = 7). (G) Macrophages from WT and Nlrx1 −/− mice were preloaded with CM-H 2 DCFDA for 30 min prior to stimulation with H. capsulatum (MOI = 10) for 20 min. Flow cytometry was performed to assess global ROS production. Data shown are the percentages of ROS + cells (left panel) and the mean fluorescence intensity (MFI) (right panel) of total live cells ( n = 4). Bars represent the mean ± SEM. ** p ≤ 0.01, *** p ≤ 0.001 [ANOVA with Bonferroni's multiple comparisons post-hoc test (A,B, G) ; 2-tailed t -test (D,E) ].

    Article Snippet: Whole cell lysates were incubated with antibodies against NLRX1 (Cell signaling), TUFM (Abcam), or rabbit IgG isotype control (GeneTex) at 4°C overnight followed by mixing with Protein A agarose beads (Merck Millipore) at 4°C for another 4 h. Lysate beads mixture was washed with IP washing buffer (0.1% NP-40 in PBS).

    Techniques: Mouse Assay, Western Blot, Expressing, Staining, Microscopy, Fluorescence, Labeling, Flow Cytometry, Cytometry

    NLRX1-TUFM is required for activation of MAPKs-AP-1 pathway and proinflammatory cytokine response to H. capsulatum . (A–C) Macrophages from WT and Nlrx1 −/− mice were stimulated with or without (0 min or medium) H. capsulatum (MOI = 5). Cell lysates were collected at 15, 30, 60, and 120 min after stimulation and subjected to Western blotting for the analysis of the indicated proteins (A) . Relative intensity of indicated protein normalized against the corresponding β-actin was shown in (B) ( n = 3). Supernatants were harvested at 18 h after stimulation, and the concentrations of TNF and IL-6 in the supernatants were quantified by ELISA ( n = 11) (C) . (D,E) Macrophages from WT mice were transfected with control siRNA or siRNA against TUFM (50 nM) for 72 h. Cells were then stimulated with or without (0 min) H. capsulatum (MOI = 5). Cell lysates were collected at 30 and 60 min after stimulation and assessed by Western blotting for the analysis of the indicated proteins (D) . Supernatants were harvested at 18 h after stimulation, and the concentrations of TNF and IL-6 in the supernatants were quantified by ELISA and are presented as the relative levels of TNF and IL-6 ( n = 6) (E) . Bars represent the mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 [2-tailed t -test (B,E) ; ANOVA with Bonferroni's multiple comparisons post-hoc test (C) ].

    Journal: Frontiers in Immunology

    Article Title: NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages

    doi: 10.3389/fimmu.2018.02761

    Figure Lengend Snippet: NLRX1-TUFM is required for activation of MAPKs-AP-1 pathway and proinflammatory cytokine response to H. capsulatum . (A–C) Macrophages from WT and Nlrx1 −/− mice were stimulated with or without (0 min or medium) H. capsulatum (MOI = 5). Cell lysates were collected at 15, 30, 60, and 120 min after stimulation and subjected to Western blotting for the analysis of the indicated proteins (A) . Relative intensity of indicated protein normalized against the corresponding β-actin was shown in (B) ( n = 3). Supernatants were harvested at 18 h after stimulation, and the concentrations of TNF and IL-6 in the supernatants were quantified by ELISA ( n = 11) (C) . (D,E) Macrophages from WT mice were transfected with control siRNA or siRNA against TUFM (50 nM) for 72 h. Cells were then stimulated with or without (0 min) H. capsulatum (MOI = 5). Cell lysates were collected at 30 and 60 min after stimulation and assessed by Western blotting for the analysis of the indicated proteins (D) . Supernatants were harvested at 18 h after stimulation, and the concentrations of TNF and IL-6 in the supernatants were quantified by ELISA and are presented as the relative levels of TNF and IL-6 ( n = 6) (E) . Bars represent the mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 [2-tailed t -test (B,E) ; ANOVA with Bonferroni's multiple comparisons post-hoc test (C) ].

    Article Snippet: Whole cell lysates were incubated with antibodies against NLRX1 (Cell signaling), TUFM (Abcam), or rabbit IgG isotype control (GeneTex) at 4°C overnight followed by mixing with Protein A agarose beads (Merck Millipore) at 4°C for another 4 h. Lysate beads mixture was washed with IP washing buffer (0.1% NP-40 in PBS).

    Techniques: Activation Assay, Mouse Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection

    NLRX1 promotes LAP through association with TUFM-ATG5-ATG12 complex. (A) Macrophages from WT mice were stimulated with or without H. capsulatum . Cell lysates were collected at 30 min after stimulation and used for immunoprecipitation with anti-NLRX1, anti-TUFM or isotype control antibodies, followed by immunoblotting with indicated antibodies. IP, IB, and WCL denote immunoprecipitation, immunoblotting, and whole-cell lysate, respectively. (B) Macrophages were stimulated with or without H. capsulatum (MOI = 5) for 60 min. Cells were fixed and stained for NLRX1 (red), TUFM (green), F-actin (violet), and nucleus compartment (blue). Cells were viewed under confocal microscope. Asterisks in the DIC/Nucleus field point to H. capsulatum yeasts. Box areas are shown at higher magnification in the bottom left corner of the corresponding image. The intensity of different fluorochromes along the white arrow in the merged image is shown as histogram on the right. (C) Macrophages from WT mice were transfected with control siRNA or siRNA against TUFM (50 nM) for 72 h. Cells were then stimulated with or without (0 min) H. capsulatum (MOI = 5) for 30 and 60 min. After stimulation, cell lysates were collected and assessed by Western blotting. (D) Macrophages from WT and Nlrx1 −/− mice were treated with or without DPI (5 μM) for 1 h prior to stimulation with H. capsulatum (MOI = 5). Cell lysates were collected after 1 h of stimulation and analyzed by Western blotting. Data shown in the lower panel of (C,D) are relative intensity of LC3-II normalized against the corresponding β-actin, mean ± SEM are shown ( n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 [ANOVA with Bonferroni's multiple comparisons post-hoc test (C,D) ].

    Journal: Frontiers in Immunology

    Article Title: NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages

    doi: 10.3389/fimmu.2018.02761

    Figure Lengend Snippet: NLRX1 promotes LAP through association with TUFM-ATG5-ATG12 complex. (A) Macrophages from WT mice were stimulated with or without H. capsulatum . Cell lysates were collected at 30 min after stimulation and used for immunoprecipitation with anti-NLRX1, anti-TUFM or isotype control antibodies, followed by immunoblotting with indicated antibodies. IP, IB, and WCL denote immunoprecipitation, immunoblotting, and whole-cell lysate, respectively. (B) Macrophages were stimulated with or without H. capsulatum (MOI = 5) for 60 min. Cells were fixed and stained for NLRX1 (red), TUFM (green), F-actin (violet), and nucleus compartment (blue). Cells were viewed under confocal microscope. Asterisks in the DIC/Nucleus field point to H. capsulatum yeasts. Box areas are shown at higher magnification in the bottom left corner of the corresponding image. The intensity of different fluorochromes along the white arrow in the merged image is shown as histogram on the right. (C) Macrophages from WT mice were transfected with control siRNA or siRNA against TUFM (50 nM) for 72 h. Cells were then stimulated with or without (0 min) H. capsulatum (MOI = 5) for 30 and 60 min. After stimulation, cell lysates were collected and assessed by Western blotting. (D) Macrophages from WT and Nlrx1 −/− mice were treated with or without DPI (5 μM) for 1 h prior to stimulation with H. capsulatum (MOI = 5). Cell lysates were collected after 1 h of stimulation and analyzed by Western blotting. Data shown in the lower panel of (C,D) are relative intensity of LC3-II normalized against the corresponding β-actin, mean ± SEM are shown ( n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 [ANOVA with Bonferroni's multiple comparisons post-hoc test (C,D) ].

    Article Snippet: Whole cell lysates were incubated with antibodies against NLRX1 (Cell signaling), TUFM (Abcam), or rabbit IgG isotype control (GeneTex) at 4°C overnight followed by mixing with Protein A agarose beads (Merck Millipore) at 4°C for another 4 h. Lysate beads mixture was washed with IP washing buffer (0.1% NP-40 in PBS).

    Techniques: Mouse Assay, Immunoprecipitation, Staining, Microscopy, Transfection, Western Blot

    Schematic model of LAP in H. capsulatum -infected macrophage. Upon macrophage encountering H. capsulatum , recognition of fungal β-glucan by Dectin-1 induces Syk phosphorylation. Activation of Syk triggers ROS production through NADPH oxidase. ROS is required for converting LC3-I to LC3-II on phagosomal membrane. Through a mechanism independent of ROS, NLRX1 promotes fungus-induced LAP through its association with TUFM that interacts with ATG5-ATG12 conjugate for LAPosome formation. Both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. The formation of LAPosome promotes downstream MAPKs and AP-1 activation, leading to production of anti-fungal proinflammatory cytokines.

    Journal: Frontiers in Immunology

    Article Title: NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages

    doi: 10.3389/fimmu.2018.02761

    Figure Lengend Snippet: Schematic model of LAP in H. capsulatum -infected macrophage. Upon macrophage encountering H. capsulatum , recognition of fungal β-glucan by Dectin-1 induces Syk phosphorylation. Activation of Syk triggers ROS production through NADPH oxidase. ROS is required for converting LC3-I to LC3-II on phagosomal membrane. Through a mechanism independent of ROS, NLRX1 promotes fungus-induced LAP through its association with TUFM that interacts with ATG5-ATG12 conjugate for LAPosome formation. Both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. The formation of LAPosome promotes downstream MAPKs and AP-1 activation, leading to production of anti-fungal proinflammatory cytokines.

    Article Snippet: Whole cell lysates were incubated with antibodies against NLRX1 (Cell signaling), TUFM (Abcam), or rabbit IgG isotype control (GeneTex) at 4°C overnight followed by mixing with Protein A agarose beads (Merck Millipore) at 4°C for another 4 h. Lysate beads mixture was washed with IP washing buffer (0.1% NP-40 in PBS).

    Techniques: Infection, Activation Assay

    Caspase-8 is required for the proteasome-mediated degradation of IRF-3. A , HT1080 cells were transfected with poly(I:C) in the absence or the presence of an inhibitor of caspase-8 (z-IETD, 10 μ m ) for the indicated times, and cell lysates were

    Journal: The Journal of Biological Chemistry

    Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *

    doi: 10.1074/jbc.M111.257022

    Figure Lengend Snippet: Caspase-8 is required for the proteasome-mediated degradation of IRF-3. A , HT1080 cells were transfected with poly(I:C) in the absence or the presence of an inhibitor of caspase-8 (z-IETD, 10 μ m ) for the indicated times, and cell lysates were

    Article Snippet: To confirm the specific involvement of caspase-8, we used ARPE19 retinal epithelial cells, which express very low levels of caspase-8 ( C ).

    Techniques: Transfection

    Caspase-8 is activated by cytosolic RIG-I-dependent signaling. A , 1080.10 cells were infected with SeV at MOI 10 for the indicated times, and the cell lysates were analyzed for the activation of caspase-8 by Western blot ( FL , full-length; CL , cleaved;

    Journal: The Journal of Biological Chemistry

    Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *

    doi: 10.1074/jbc.M111.257022

    Figure Lengend Snippet: Caspase-8 is activated by cytosolic RIG-I-dependent signaling. A , 1080.10 cells were infected with SeV at MOI 10 for the indicated times, and the cell lysates were analyzed for the activation of caspase-8 by Western blot ( FL , full-length; CL , cleaved;

    Article Snippet: To confirm the specific involvement of caspase-8, we used ARPE19 retinal epithelial cells, which express very low levels of caspase-8 ( C ).

    Techniques: Infection, Activation Assay, Western Blot

    Caspase-8 activity is essential for the cleavage of IRF-3. A , P2.1 cells expressing IRF-3, were pretreated with the inhibitors of multiple caspases (Z, z-VAD, 10 μ m ), caspase-1 (z-WEHD, 10 μ m ), or caspase-8 (z-IETD, 10 μ m ) for

    Journal: The Journal of Biological Chemistry

    Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *

    doi: 10.1074/jbc.M111.257022

    Figure Lengend Snippet: Caspase-8 activity is essential for the cleavage of IRF-3. A , P2.1 cells expressing IRF-3, were pretreated with the inhibitors of multiple caspases (Z, z-VAD, 10 μ m ), caspase-1 (z-WEHD, 10 μ m ), or caspase-8 (z-IETD, 10 μ m ) for

    Article Snippet: To confirm the specific involvement of caspase-8, we used ARPE19 retinal epithelial cells, which express very low levels of caspase-8 ( C ).

    Techniques: Activity Assay, Expressing

    Caspase-8-mediated cleavage of IRF-3 is an intermediate step in its proteasome-mediated degradation. IRF-3 undergoes proteasome-mediated degradation in response to dsRNA-dependent signaling. Stimulation of TLR3 or RIG-I signaling by dsRNA activates caspase-8

    Journal: The Journal of Biological Chemistry

    Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *

    doi: 10.1074/jbc.M111.257022

    Figure Lengend Snippet: Caspase-8-mediated cleavage of IRF-3 is an intermediate step in its proteasome-mediated degradation. IRF-3 undergoes proteasome-mediated degradation in response to dsRNA-dependent signaling. Stimulation of TLR3 or RIG-I signaling by dsRNA activates caspase-8

    Article Snippet: To confirm the specific involvement of caspase-8, we used ARPE19 retinal epithelial cells, which express very low levels of caspase-8 ( C ).

    Techniques:

    Mutation of a caspase-8 recognition motif leads to impaired cleavage and degradation of IRF-3. P2.1 cells expressing Wt or the D121E ( DE ) mutant of IRF-3 were used in these experiments ( A–C ). A , cells were transfected with poly(I:C); cell lysates

    Journal: The Journal of Biological Chemistry

    Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *

    doi: 10.1074/jbc.M111.257022

    Figure Lengend Snippet: Mutation of a caspase-8 recognition motif leads to impaired cleavage and degradation of IRF-3. P2.1 cells expressing Wt or the D121E ( DE ) mutant of IRF-3 were used in these experiments ( A–C ). A , cells were transfected with poly(I:C); cell lysates

    Article Snippet: To confirm the specific involvement of caspase-8, we used ARPE19 retinal epithelial cells, which express very low levels of caspase-8 ( C ).

    Techniques: Mutagenesis, Expressing, Transfection

    TLR7 triggering leads to tyrosine kinase dependent Src K63-linked ubiquitination and TNFR-associated factor 6 (TRAF6) tyrosine phosphorylation. hTLR7-HEK293 cells, transiently transfected with plasmids coding for hemagglutinin (HA)-tagged K63-only, were pretreated or not with PP2 (20 µM) for 30 min and stimulated for 2 h with R848 (10 µM). (A) Total cell lysates were immunoprecipitated with anti-pSFKs antibody and immunoblotted using anti-HA antibody for ubiquitin detection. After stripping, the presence of p-SFKs was assessed by immunoblot analysis using specific antibodies, as control. (B) Total cell lysates were immunoprecipitated with anti-TRAF6 and immunoblotted with antibodies against pSFKs, Src, and pTyr. (C) TRAF6-specific immunoprecipitates were eluted with 2% SDS in order to disrupt the TRAF6-SFKs interaction and after dilution with PBS re-immunoprecipitated with anti-TRAF6 antibody. Results are representative of three independent experiments. Histograms on the right side of the blots show the quantification of the pulled-down proteins, calculated at least on two independent experiments. Each signal was normalized to the signal of the protein used for the pull-down and expressed as fold induction (mean ± SD) compared to untreated sample. *** P

    Journal: Frontiers in Immunology

    Article Title: Src Family Kinases Regulate Interferon Regulatory Factor 1 K63 Ubiquitination following Activation by TLR7/8 Vaccine Adjuvant in Human Monocytes and B Cells

    doi: 10.3389/fimmu.2018.00330

    Figure Lengend Snippet: TLR7 triggering leads to tyrosine kinase dependent Src K63-linked ubiquitination and TNFR-associated factor 6 (TRAF6) tyrosine phosphorylation. hTLR7-HEK293 cells, transiently transfected with plasmids coding for hemagglutinin (HA)-tagged K63-only, were pretreated or not with PP2 (20 µM) for 30 min and stimulated for 2 h with R848 (10 µM). (A) Total cell lysates were immunoprecipitated with anti-pSFKs antibody and immunoblotted using anti-HA antibody for ubiquitin detection. After stripping, the presence of p-SFKs was assessed by immunoblot analysis using specific antibodies, as control. (B) Total cell lysates were immunoprecipitated with anti-TRAF6 and immunoblotted with antibodies against pSFKs, Src, and pTyr. (C) TRAF6-specific immunoprecipitates were eluted with 2% SDS in order to disrupt the TRAF6-SFKs interaction and after dilution with PBS re-immunoprecipitated with anti-TRAF6 antibody. Results are representative of three independent experiments. Histograms on the right side of the blots show the quantification of the pulled-down proteins, calculated at least on two independent experiments. Each signal was normalized to the signal of the protein used for the pull-down and expressed as fold induction (mean ± SD) compared to untreated sample. *** P

    Article Snippet: For immunoblot and immunoprecipitation analysis, we used the following antibodies: anti-IRF-1 (cat# 8478 Cell Signaling for immunoblot and cat# sc-497 Santa Cruz Biotechnology for immunoprecipitation); anti-TRAF6 (cat# 8028 Cell Signaling); anti-Src (cat# 2108 Cell Signaling); anti-p-SFKs (Tyr416) (2101 Cell Signaling); anti-p-Tyrosine (cat# 8954 Cell Signaling); anti-HA (cat# 2367 Cell Signaling); anti-cIAP2 (cat# ab32059 Abcam), anti-β-actin (cat# ab8227 Abcam); and HRP-conjugated secondary antibodies (DAKO).

    Techniques: Transfection, Immunoprecipitation, Stripping Membranes

    Inhibition of SFKs impairs the stability of the SFKs-TRAF6-IRF-1-cIAP2 complex (A–C) hTLR7-HEK293 cells were pretreated or not with PP2 (20 µM) for 30 min and stimulated for 2 h with R848 (10 µM). Total cell lysates were immunoprecipitated using an anti-TRAF6 (A) , anti-interferon regulatory factor 1 (IRF-1) (B) , or anti-pSFKs (C) antibody, respectively. Co-immunoprecipitated cIAP2, TNFR-associated factor 6 (TRAF6), SFKs, and IRF-1 proteins were detected by immunoblot. Histograms on the right side of the blots show the quantification of the relative amount of the pulled-down proteins, calculated on three independent experiments. Each signal was normalized to the signal of the protein used for the pull-down and expressed as fold induction (mean ± SD) compared to untreated sample. *** P

    Journal: Frontiers in Immunology

    Article Title: Src Family Kinases Regulate Interferon Regulatory Factor 1 K63 Ubiquitination following Activation by TLR7/8 Vaccine Adjuvant in Human Monocytes and B Cells

    doi: 10.3389/fimmu.2018.00330

    Figure Lengend Snippet: Inhibition of SFKs impairs the stability of the SFKs-TRAF6-IRF-1-cIAP2 complex (A–C) hTLR7-HEK293 cells were pretreated or not with PP2 (20 µM) for 30 min and stimulated for 2 h with R848 (10 µM). Total cell lysates were immunoprecipitated using an anti-TRAF6 (A) , anti-interferon regulatory factor 1 (IRF-1) (B) , or anti-pSFKs (C) antibody, respectively. Co-immunoprecipitated cIAP2, TNFR-associated factor 6 (TRAF6), SFKs, and IRF-1 proteins were detected by immunoblot. Histograms on the right side of the blots show the quantification of the relative amount of the pulled-down proteins, calculated on three independent experiments. Each signal was normalized to the signal of the protein used for the pull-down and expressed as fold induction (mean ± SD) compared to untreated sample. *** P

    Article Snippet: For immunoblot and immunoprecipitation analysis, we used the following antibodies: anti-IRF-1 (cat# 8478 Cell Signaling for immunoblot and cat# sc-497 Santa Cruz Biotechnology for immunoprecipitation); anti-TRAF6 (cat# 8028 Cell Signaling); anti-Src (cat# 2108 Cell Signaling); anti-p-SFKs (Tyr416) (2101 Cell Signaling); anti-p-Tyrosine (cat# 8954 Cell Signaling); anti-HA (cat# 2367 Cell Signaling); anti-cIAP2 (cat# ab32059 Abcam), anti-β-actin (cat# ab8227 Abcam); and HRP-conjugated secondary antibodies (DAKO).

    Techniques: Inhibition, Immunoprecipitation

    Schematic illustration of SFKs mechanism of action in interferon regulatory factor 1 (IRF-1) K63-Ubiquitination. (A) Activation of the TLR7/8 pathway promotes the interaction between SFKs and TNFR-associated factor 6 (TRAF6) allowing the recruitment of both cIAP2 and IRF-1 by TRAF6 with the subsequent K63-ubiquitination and accumulation of IRF-1. (B) Preincubation with PP2 impairs the capacity of SFKs to interact with TRAF6 and the formation of the TRAF6-IRF1-cIAP2 complex, inhibiting K63-ubiquitination of IRF-1, which is, however, K48-ubiquitinylated and it is consequently degraded via proteasome.

    Journal: Frontiers in Immunology

    Article Title: Src Family Kinases Regulate Interferon Regulatory Factor 1 K63 Ubiquitination following Activation by TLR7/8 Vaccine Adjuvant in Human Monocytes and B Cells

    doi: 10.3389/fimmu.2018.00330

    Figure Lengend Snippet: Schematic illustration of SFKs mechanism of action in interferon regulatory factor 1 (IRF-1) K63-Ubiquitination. (A) Activation of the TLR7/8 pathway promotes the interaction between SFKs and TNFR-associated factor 6 (TRAF6) allowing the recruitment of both cIAP2 and IRF-1 by TRAF6 with the subsequent K63-ubiquitination and accumulation of IRF-1. (B) Preincubation with PP2 impairs the capacity of SFKs to interact with TRAF6 and the formation of the TRAF6-IRF1-cIAP2 complex, inhibiting K63-ubiquitination of IRF-1, which is, however, K48-ubiquitinylated and it is consequently degraded via proteasome.

    Article Snippet: For immunoblot and immunoprecipitation analysis, we used the following antibodies: anti-IRF-1 (cat# 8478 Cell Signaling for immunoblot and cat# sc-497 Santa Cruz Biotechnology for immunoprecipitation); anti-TRAF6 (cat# 8028 Cell Signaling); anti-Src (cat# 2108 Cell Signaling); anti-p-SFKs (Tyr416) (2101 Cell Signaling); anti-p-Tyrosine (cat# 8954 Cell Signaling); anti-HA (cat# 2367 Cell Signaling); anti-cIAP2 (cat# ab32059 Abcam), anti-β-actin (cat# ab8227 Abcam); and HRP-conjugated secondary antibodies (DAKO).

    Techniques: Activation Assay

    A positive feedback loop comprised of Foxo1-BLNK-p38 that regulates pre-B cell differentiation

    Journal: Nature immunology

    Article Title: A self-reinforcing regulatory network triggered by limiting interleukin-7 activates pre-BCR signaling and differentiation

    doi: 10.1038/ni.2210

    Figure Lengend Snippet: A positive feedback loop comprised of Foxo1-BLNK-p38 that regulates pre-B cell differentiation

    Article Snippet: Antibodies used to probe the protein blots were; anti-Foxo1 (Cell Signaling, #9462), anti-Foxo3a (Cell Signaling, #9467), anti-Foxo4 (Cell Signaling, #9472), anti-p-Foxo1 (Cell Signaling, #9461), anti-p-Foxo3a (Cell Signaling, #9466), anti-p-Akt (Cell Signaling, #4058), anti-Akt (Cell Signaling, #9272), anti-p-p38 (Cell Signaling, #9211), anti-p-38 (Cell Signaling, #9212), anti-Pax5 (Santa Cruz, sc-1974), anti-BLNK (Santa Cruz, sc-15345), anti-Syk (Santa Cruz, sc-1077), anti-IRF-4 (Santa Cruz, sc-6059), anti-IRF-8 (sc-6058), anti-HPRT (Santa Cruz, sc-20975).

    Techniques: Cell Differentiation

    ChIPseq analysis of the Foxo1 cistrome in IRF-4,8 −/− pre-B cells

    Journal: Nature immunology

    Article Title: A self-reinforcing regulatory network triggered by limiting interleukin-7 activates pre-BCR signaling and differentiation

    doi: 10.1038/ni.2210

    Figure Lengend Snippet: ChIPseq analysis of the Foxo1 cistrome in IRF-4,8 −/− pre-B cells

    Article Snippet: Antibodies used to probe the protein blots were; anti-Foxo1 (Cell Signaling, #9462), anti-Foxo3a (Cell Signaling, #9467), anti-Foxo4 (Cell Signaling, #9472), anti-p-Foxo1 (Cell Signaling, #9461), anti-p-Foxo3a (Cell Signaling, #9466), anti-p-Akt (Cell Signaling, #4058), anti-Akt (Cell Signaling, #9272), anti-p-p38 (Cell Signaling, #9211), anti-p-38 (Cell Signaling, #9212), anti-Pax5 (Santa Cruz, sc-1974), anti-BLNK (Santa Cruz, sc-15345), anti-Syk (Santa Cruz, sc-1077), anti-IRF-4 (Santa Cruz, sc-6059), anti-IRF-8 (sc-6058), anti-HPRT (Santa Cruz, sc-20975).

    Techniques:

    Foxo1 is necessary for inducing two key components of pre-BCR signaling

    Journal: Nature immunology

    Article Title: A self-reinforcing regulatory network triggered by limiting interleukin-7 activates pre-BCR signaling and differentiation

    doi: 10.1038/ni.2210

    Figure Lengend Snippet: Foxo1 is necessary for inducing two key components of pre-BCR signaling

    Article Snippet: Antibodies used to probe the protein blots were; anti-Foxo1 (Cell Signaling, #9462), anti-Foxo3a (Cell Signaling, #9467), anti-Foxo4 (Cell Signaling, #9472), anti-p-Foxo1 (Cell Signaling, #9461), anti-p-Foxo3a (Cell Signaling, #9466), anti-p-Akt (Cell Signaling, #4058), anti-Akt (Cell Signaling, #9272), anti-p-p38 (Cell Signaling, #9211), anti-p-38 (Cell Signaling, #9212), anti-Pax5 (Santa Cruz, sc-1974), anti-BLNK (Santa Cruz, sc-15345), anti-Syk (Santa Cruz, sc-1077), anti-IRF-4 (Santa Cruz, sc-6059), anti-IRF-8 (sc-6058), anti-HPRT (Santa Cruz, sc-20975).

    Techniques:

    Foxo1 and Pax5 cooperate to induce Blnk expression upon attenuation of IL-7 signaling

    Journal: Nature immunology

    Article Title: A self-reinforcing regulatory network triggered by limiting interleukin-7 activates pre-BCR signaling and differentiation

    doi: 10.1038/ni.2210

    Figure Lengend Snippet: Foxo1 and Pax5 cooperate to induce Blnk expression upon attenuation of IL-7 signaling

    Article Snippet: Antibodies used to probe the protein blots were; anti-Foxo1 (Cell Signaling, #9462), anti-Foxo3a (Cell Signaling, #9467), anti-Foxo4 (Cell Signaling, #9472), anti-p-Foxo1 (Cell Signaling, #9461), anti-p-Foxo3a (Cell Signaling, #9466), anti-p-Akt (Cell Signaling, #4058), anti-Akt (Cell Signaling, #9272), anti-p-p38 (Cell Signaling, #9211), anti-p-38 (Cell Signaling, #9212), anti-Pax5 (Santa Cruz, sc-1974), anti-BLNK (Santa Cruz, sc-15345), anti-Syk (Santa Cruz, sc-1077), anti-IRF-4 (Santa Cruz, sc-6059), anti-IRF-8 (sc-6058), anti-HPRT (Santa Cruz, sc-20975).

    Techniques: Expressing

    Activation of the Foxo1-BLNK-p38 module is specific to pre-B cells

    Journal: Nature immunology

    Article Title: A self-reinforcing regulatory network triggered by limiting interleukin-7 activates pre-BCR signaling and differentiation

    doi: 10.1038/ni.2210

    Figure Lengend Snippet: Activation of the Foxo1-BLNK-p38 module is specific to pre-B cells

    Article Snippet: Antibodies used to probe the protein blots were; anti-Foxo1 (Cell Signaling, #9462), anti-Foxo3a (Cell Signaling, #9467), anti-Foxo4 (Cell Signaling, #9472), anti-p-Foxo1 (Cell Signaling, #9461), anti-p-Foxo3a (Cell Signaling, #9466), anti-p-Akt (Cell Signaling, #4058), anti-Akt (Cell Signaling, #9272), anti-p-p38 (Cell Signaling, #9211), anti-p-38 (Cell Signaling, #9212), anti-Pax5 (Santa Cruz, sc-1974), anti-BLNK (Santa Cruz, sc-15345), anti-Syk (Santa Cruz, sc-1077), anti-IRF-4 (Santa Cruz, sc-6059), anti-IRF-8 (sc-6058), anti-HPRT (Santa Cruz, sc-20975).

    Techniques: Activation Assay

    SAHA-induced acetylation of p53 led to dissociation from MDM2, contributing to cell apoptosis.

    Journal: The Journal of Clinical Investigation

    Article Title: Efficacious proteasome/HDAC inhibitor combination therapy for primary effusion lymphoma

    doi: 10.1172/JCI64503

    Figure Lengend Snippet: SAHA-induced acetylation of p53 led to dissociation from MDM2, contributing to cell apoptosis.

    Article Snippet: Primary antibodies to Bax, Bcl-XL , c-MYC, p21, P-IκB-α, caspase-8, acetyl-H3, total H3, acetyl p53 (Lys-382), phospho-p53 (Ser-15), GRP-78, GRP-94, P-eIF2α, total eIF2α, and CHOP were from Cell Signaling Technology; GAPDH, β-actin, IRF-4, c-FLIP, ubiquitin, and p53 were from Santa Cruz Biotechnology Inc.; total IκB-α, Bcl-2, CD30, and annexin V–FITC were from BD Pharmingen; ATF-6 was from Imgenex; and cIAP2 was from Abcam.

    Techniques:

    Btz downregulates c-MYC and stabilizes the expression of phosphorylated p53 and γH2AX and downstream p53 targets p21 and Bax in PEL xenografts.

    Journal: The Journal of Clinical Investigation

    Article Title: Efficacious proteasome/HDAC inhibitor combination therapy for primary effusion lymphoma

    doi: 10.1172/JCI64503

    Figure Lengend Snippet: Btz downregulates c-MYC and stabilizes the expression of phosphorylated p53 and γH2AX and downstream p53 targets p21 and Bax in PEL xenografts.

    Article Snippet: Primary antibodies to Bax, Bcl-XL , c-MYC, p21, P-IκB-α, caspase-8, acetyl-H3, total H3, acetyl p53 (Lys-382), phospho-p53 (Ser-15), GRP-78, GRP-94, P-eIF2α, total eIF2α, and CHOP were from Cell Signaling Technology; GAPDH, β-actin, IRF-4, c-FLIP, ubiquitin, and p53 were from Santa Cruz Biotechnology Inc.; total IκB-α, Bcl-2, CD30, and annexin V–FITC were from BD Pharmingen; ATF-6 was from Imgenex; and cIAP2 was from Abcam.

    Techniques: Expressing

    TLR4 is a direct target of miR-140-5p. (A) Schematic demonstration of the potential binding sites (Binding site 1 was conserved, while binding site 2 and 3 were poorly conserved) for miR-140-5p in the 3′UTR of TLR4. (B) Dual luciferase reporter assay. pmirGLO-TLR4-3′UTR-1 contained binding site 1 and pmirGLO-TLR4-3′UTR-2 contained binding sites 2 and 3. TLR4 protein expression due to the (C) overexpression and (D) inhibition of miR-140-5p was analyzed in BEAS-2B cells. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Decreased microRNA-140-5p contributes to respiratory syncytial virus disease through targeting Toll-like receptor 4

    doi: 10.3892/etm.2018.6272

    Figure Lengend Snippet: TLR4 is a direct target of miR-140-5p. (A) Schematic demonstration of the potential binding sites (Binding site 1 was conserved, while binding site 2 and 3 were poorly conserved) for miR-140-5p in the 3′UTR of TLR4. (B) Dual luciferase reporter assay. pmirGLO-TLR4-3′UTR-1 contained binding site 1 and pmirGLO-TLR4-3′UTR-2 contained binding sites 2 and 3. TLR4 protein expression due to the (C) overexpression and (D) inhibition of miR-140-5p was analyzed in BEAS-2B cells. *P

    Article Snippet: Following three washes with PBS with Tween 20 (5 min/wash), the membranes were incubated with the following primary antibodies at 4°C overnight: TLR4 (cat no. 14358; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) and GAPDH (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc.).

    Techniques: Binding Assay, Luciferase, Reporter Assay, Expressing, Over Expression, Inhibition

    SAHA-induced acetylation of p53 led to dissociation from MDM2, contributing to cell apoptosis.

    Journal: The Journal of Clinical Investigation

    Article Title: Efficacious proteasome/HDAC inhibitor combination therapy for primary effusion lymphoma

    doi: 10.1172/JCI64503

    Figure Lengend Snippet: SAHA-induced acetylation of p53 led to dissociation from MDM2, contributing to cell apoptosis.

    Article Snippet: Primary antibodies to Bax, Bcl-XL , c-MYC, p21, P-IκB-α, caspase-8, acetyl-H3, total H3, acetyl p53 (Lys-382), phospho-p53 (Ser-15), GRP-78, GRP-94, P-eIF2α, total eIF2α, and CHOP were from Cell Signaling Technology; GAPDH, β-actin, IRF-4, c-FLIP, ubiquitin, and p53 were from Santa Cruz Biotechnology Inc.; total IκB-α, Bcl-2, CD30, and annexin V–FITC were from BD Pharmingen; ATF-6 was from Imgenex; and cIAP2 was from Abcam.

    Techniques:

    Btz downregulates c-MYC and stabilizes the expression of phosphorylated p53 and γH2AX and downstream p53 targets p21 and Bax in PEL xenografts.

    Journal: The Journal of Clinical Investigation

    Article Title: Efficacious proteasome/HDAC inhibitor combination therapy for primary effusion lymphoma

    doi: 10.1172/JCI64503

    Figure Lengend Snippet: Btz downregulates c-MYC and stabilizes the expression of phosphorylated p53 and γH2AX and downstream p53 targets p21 and Bax in PEL xenografts.

    Article Snippet: Primary antibodies to Bax, Bcl-XL , c-MYC, p21, P-IκB-α, caspase-8, acetyl-H3, total H3, acetyl p53 (Lys-382), phospho-p53 (Ser-15), GRP-78, GRP-94, P-eIF2α, total eIF2α, and CHOP were from Cell Signaling Technology; GAPDH, β-actin, IRF-4, c-FLIP, ubiquitin, and p53 were from Santa Cruz Biotechnology Inc.; total IκB-α, Bcl-2, CD30, and annexin V–FITC were from BD Pharmingen; ATF-6 was from Imgenex; and cIAP2 was from Abcam.

    Techniques: Expressing