irf8-mediated inhibition Search Results


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  • 98
    ATCC sendai virus
    Sendai Virus, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore ezh2
    <t>EZH2</t> directly silences IRF8 expression
    Ezh2, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 911 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Epitope Biotech virf 3
    <t>vIRF-3</t> represses IRF-5-mediated transactivation of the p21 promoter in H1299 cells. H1299 cells were transfected with a p21 luciferase reporter construct and the constructs given in the table below . Luciferase activity was measured 48 h after transfection.
    Virf 3, supplied by Epitope Biotech, used in various techniques. Bioz Stars score: 89/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc akt
    Effect of HQ on <t>AKT</t> activation in IRF3 pathway (A) RAW264.7 cells (1×10 6 cells/ml) pretreated with either HQ (50 µM) or vehicle for 30 min were incubated with LPS (1.0 µg/ml) for the indicated time. Phosphorylated and total levels of TBK1 and AKT in total cell lysates were determined by Western blot analysis. (B) RAW264.7 cells (1×10 6 cells/ml) transfected with HA-AKT (1.0 µg/ml) for 24 h were treated with the indicated doses of HQ for another 24 h, and phosphorylated and total levels of <t>IRF-3</t> and HA in total cell lysates were determined by Western blot analysis. (C) RAW264.7 cells (1×10 6 cells/ml) were transfected with either HA-AKT (1.0 µg/ml) or HA-AKT C310A (1.0 µg/ml) for 48 h. Phosphorylated and total levels of IRF-3, AKT, and HA in total cell lysates were determined by Western blot analysis. (D) RAW264.7 cells (1×10 6 cells/ml) transfected with either HA-AKT1 (1.0 µg/ ml), HA-AKT2 (1.0 µg/ml), or HA-AKT3 (1.0 µg/ml) for 24 h were treated with either HQ (50 µM) or vehicle for another 24 h. Phosphorylated and total levels of IRF-3, AKT, and HA in the total cell lysates were determined by Western blot analysis. (E) HEK293 cells (1×10 6 cells/ml) cotransfected with TBK1 (1.0 µg/ml), IRF-3-Luc (1.0 µg/ml), β-gal (0.1 µg/ml), and HA-AKT1 (1.0 µg/ml), HA-AKT2 (1.0 µg/ml), HA-AKT3 (1.0 µg/ml), or HA-AKT KD (kinase domain deletion mutant) for 24 h were treated with either HQ (50 µM) or vehicle for another 24 h. Luciferase activity was measured with a luminometer. All luciferase reporter gene activities were normalized to β-galactosidase activity. * p
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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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    99
    Active Motif ezh2
    Inhibition of <t>EZH2</t> prevents repression of OCL negative factors and inhibits OCL formation. BMM cells were cultured with MCSF for 3 days (d-3 to d0) to form OCLp before addition of RANKL/MCSF to generate OCL. (A) OCLp were cultured with RANKL/MCSF in the presence of the indicated concentrations of GSK126 for 5 days. TRAP+ OCL formed were analyzed by microscopy (representative images on left) and the counts for multinucleated (n≥3) TRAP+ OCL/well graphed (right). (B) GSK126 (10 μM) was added at the indicated time intervals during the processes of OCLp and OCL (d4) formation and representative images and counts for multinucleated (n≥3) TRAP+ OCL/well are shown. (C) OCLp (d0) or stimulated with RANKL/MCSF +/− GSK126 (10 μM; GSK) for the indicated times were analyzed for Ezh2 mRNA by qPCR. (D) OCLp (d0) or stimulated with RANKL/MCSF for the indicated times were analyzed for total cellular levels of EZH2, H3K27me3, total H3 and βactin by WB. (E) OCLp cultures (d0) or after stimulation with RANKL/MCSF +/− GSK126 (10 μM) for 1 day were analyzed for total cellular levels of EZH2, H3K27me3, H3K9ac, H3 and βactin by WB. (F) OCLp (d0) or after stimulation with RANKL/MCSF +/− GSK126 (10 μM) for 1 or 5 days were analyzed for the expression of OCL-specific genes NFATc1 , RANK , CatK , DC-STAMP by qPCR. (G) OCLp (d0) or after stimulation with RANKL/MCSF +/− GSK126 (10 μM) for 1 day were analyzed for the expression of OCL-negative regulators MafB , Irf8 , Arg1 , and Bcl6 by qPCR. WB analyses show representative of 3 independent experiments and error bars in mRNA experiments represent SEM for 4-17 biological replicates. Statistical analysis used one-way ANOVA with Dunnett’s multiple comparisons test comparing to no GSK126 control (B), 0 time (C), or with Tukay’s multiple comparisons test for all means (G), and two-way ANOVA with Sidak multiple comparisons for day and treatment (F). Note that in C, d1 and d5 were not found to be significantly different from time 0.
    Ezh2, supplied by Active Motif, used in various techniques. Bioz Stars score: 99/100, based on 939 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti irf 1
    Src family kinases are required for interferon regulatory factor 1 <t>(IRF-1)</t> protein accumulation in response to TLR7/8 stimulation in monocytes and B cells. (A,C) Immunoblot analysis of IRF-1 and p-SFKs in THP-1 (A) and primary B cells (C) pretreated with PP2 (20 µM) or DMSO alone and stimulated with R848 (10 µM) for 2 h. After stripping, filters were re-probed with an antibody against actin as loading control. Results are representative at least of three independent experiments. The histograms on the right of the blot show the results of the densitometric analysis on three independent experiments. Each signal was normalized to its respective actin signal on the same blot and expressed as fold induction (mean ± SD) compared to untreated sample. (B,D) qRT-PCR analysis of lRF-1 mRNA in THP-1 (B) and human B cells (D) pretreated with PP2 (20 µM) or DMSO alone and stimulated with R848 (10 µM) for 2 h. The relative abundance of the gene transcripts was determined on triplicate samples from at least three independent experiments using the ΔΔCt method and is expressed as the normalized fold expression (mean ± SD) compared to untreated sample. ** P
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    90
    Lifespan Biosciences hmx3
    Regulation of HMX2 and <t>HMX3</t> by KMT2A and ERK-signalling. (A) RQ-PCR analysis of EOL-1 (left) and MV4-11 (middle) treated for siRNA-mediated knockdown of KMT2A demonstrates raised expression levels of HMX2 and HMX3, indicating a repressive impact of KMT2A. This treatment resulted in enhanced expression of HMX3 in HL-60 cells which did not express HMX2 (right). Asterisks indicate calculated p-values obtained by t-Test analysis of controls (siCTR) and siRNA-targeted knockdown. (B) RQ-PCR analysis of KMT2A of EOL-1 cells treated for siRNA-mediated knockdown of HMX2 (left) and HMX3 (right) showed unaltered KMT2A expression levels, discounting any regulatory impact. (C) Quantification of KMT2A transcripts in AML cell lines and primary granulocytes showed low expression levels in EOL-1, MV4-11 and in eosinophils. (D) RQ-PCR analysis of HMX2 showed elevated expression levels in EOL-1 cells treated with PDGFRA-inhibitor dasatinib. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (DMSO) and pharmacological treatments. (E) Treatment of EOL-1 cells with ERK-inhbitor PD98059 resulted in reduced levels of phospho-ERK as analyzed by Western blot (left), and in elevated transcript levels of HMX2 (middle) and HMX3 (right) as analyzed by RQ-PCR. (F) Treatment of EOL-1 cells with ERK-activator FLT3LG resulted in elevated levels of phosphorylated ERK (above) and in reduced transcript levels of HMX2 and HMX3 (below).
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    93
    Cell Signaling Technology Inc irf 1
    ISRE induction by ChX710 is dependent on MAVS and <t>IRF1.</t> ( a ) MAVS was silenced by siRNA transfection in ISRE-luciferase reporter cells. After 48 hours of cultures, cells were stimulated with ChX710. After 24 hours of incubation, luciferase induction was determined. ( b ) Same experiment as ( a ) using STING-specific siRNA. ( c ) Same experiment as ( a ) using IRF1-specific siRNA. ( d ) Same experiment as ( a ) using IRF3-specific siRNA. ( e,f ) IRF3 phosphorylation was determined by western-blot in HEK-293T ( e ) or A549 cells ( f ) treated for 24 hours with DMSO alone or ChX710 at 25 μM or 50 μM. ( g ) Schematic model of the signaling pathways induced by ChX710. IRF3 phosphorylation is induced, but this is insufficient to activate IFN-β expression. Data represent means ± SD of three independent experiments, except for ( e ) and ( f ) that corresponds to representative results. ** P
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    Durect Corporation 1002 osmotic minipump
    ISRE induction by ChX710 is dependent on MAVS and <t>IRF1.</t> ( a ) MAVS was silenced by siRNA transfection in ISRE-luciferase reporter cells. After 48 hours of cultures, cells were stimulated with ChX710. After 24 hours of incubation, luciferase induction was determined. ( b ) Same experiment as ( a ) using STING-specific siRNA. ( c ) Same experiment as ( a ) using IRF1-specific siRNA. ( d ) Same experiment as ( a ) using IRF3-specific siRNA. ( e,f ) IRF3 phosphorylation was determined by western-blot in HEK-293T ( e ) or A549 cells ( f ) treated for 24 hours with DMSO alone or ChX710 at 25 μM or 50 μM. ( g ) Schematic model of the signaling pathways induced by ChX710. IRF3 phosphorylation is induced, but this is insufficient to activate IFN-β expression. Data represent means ± SD of three independent experiments, except for ( e ) and ( f ) that corresponds to representative results. ** P
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    Cell Signaling Technology Inc antibodies against 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) ].
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    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
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    MathWorks Inc matlab r2010a
    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
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    Novartis nvp bez235
    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
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    cd200  (Abcam)
    91
    Abcam cd200
    <t>CD200</t> antibody blockade limits tumor progression in GEM model of PDAC. (A) Representative immunofluorescence from pancreatic tissue of KPC-Brca2 mice stained for DAPI (blue), a-SMA (green), and CD200 (red). (B) KPC-Brca2 mice were treated at 6 weeks of age with 200 µg (intraperitoneal injection three times/week) of isotype control or CD200 antibodies (n=5 mice/group). (C) Histology was pathologically scored for PanIN lesions and quantified. Mean±SD; *p
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    Cell Signaling Technology Inc rabbit anti human phospho akt ser473
    <t>CD200</t> antibody blockade limits tumor progression in GEM model of PDAC. (A) Representative immunofluorescence from pancreatic tissue of KPC-Brca2 mice stained for DAPI (blue), a-SMA (green), and CD200 (red). (B) KPC-Brca2 mice were treated at 6 weeks of age with 200 µg (intraperitoneal injection three times/week) of isotype control or CD200 antibodies (n=5 mice/group). (C) Histology was pathologically scored for PanIN lesions and quantified. Mean±SD; *p
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    Cell Signaling Technology Inc phospho akt
    <t>CD200</t> antibody blockade limits tumor progression in GEM model of PDAC. (A) Representative immunofluorescence from pancreatic tissue of KPC-Brca2 mice stained for DAPI (blue), a-SMA (green), and CD200 (red). (B) KPC-Brca2 mice were treated at 6 weeks of age with 200 µg (intraperitoneal injection three times/week) of isotype control or CD200 antibodies (n=5 mice/group). (C) Histology was pathologically scored for PanIN lesions and quantified. Mean±SD; *p
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    Cell Signaling Technology Inc phospho rip1 s166
    <t>CD200</t> antibody blockade limits tumor progression in GEM model of PDAC. (A) Representative immunofluorescence from pancreatic tissue of KPC-Brca2 mice stained for DAPI (blue), a-SMA (green), and CD200 (red). (B) KPC-Brca2 mice were treated at 6 weeks of age with 200 µg (intraperitoneal injection three times/week) of isotype control or CD200 antibodies (n=5 mice/group). (C) Histology was pathologically scored for PanIN lesions and quantified. Mean±SD; *p
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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

    vIRF-3 represses IRF-5-mediated transactivation of the p21 promoter in H1299 cells. H1299 cells were transfected with a p21 luciferase reporter construct and the constructs given in the table below . Luciferase activity was measured 48 h after transfection.

    Journal:

    Article Title: The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 *The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 * S⃞

    doi: 10.1074/jbc.M809252200

    Figure Lengend Snippet: vIRF-3 represses IRF-5-mediated transactivation of the p21 promoter in H1299 cells. H1299 cells were transfected with a p21 luciferase reporter construct and the constructs given in the table below . Luciferase activity was measured 48 h after transfection.

    Article Snippet: In addition, in vivo binding of IRF-5 to relevant target gene promoters and their activation was reduced in the presence of vIRF-3 ( ).

    Techniques: Transfection, Luciferase, Construct, Activity Assay

    Inhibition of IRF-5 DNA binding by vIRF-3. A , inhibition of binding to an ISRE element. Oligonucleotide pull-down was carried out with a biotinylated ISRE element as probe. Probe ( lanes 1, 3, 5, 7 , and 8 ) or mutated probe ( lanes 2, 4 , and 6 ) were

    Journal:

    Article Title: The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 *The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 * S⃞

    doi: 10.1074/jbc.M809252200

    Figure Lengend Snippet: Inhibition of IRF-5 DNA binding by vIRF-3. A , inhibition of binding to an ISRE element. Oligonucleotide pull-down was carried out with a biotinylated ISRE element as probe. Probe ( lanes 1, 3, 5, 7 , and 8 ) or mutated probe ( lanes 2, 4 , and 6 ) were

    Article Snippet: In addition, in vivo binding of IRF-5 to relevant target gene promoters and their activation was reduced in the presence of vIRF-3 ( ).

    Techniques: Inhibition, Binding Assay

    Subcellular localization of vIRF-3 and IRF-5. A , separation of nuclear and cytoplasmic proteins was carried out with one million KSHV-positive cells (BC-3 and JSC-1) as well as KSHV-negative B-cells (Akata and BJAB). IRF-5 was detectable in both cytoplasmic

    Journal:

    Article Title: The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 *The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 * S⃞

    doi: 10.1074/jbc.M809252200

    Figure Lengend Snippet: Subcellular localization of vIRF-3 and IRF-5. A , separation of nuclear and cytoplasmic proteins was carried out with one million KSHV-positive cells (BC-3 and JSC-1) as well as KSHV-negative B-cells (Akata and BJAB). IRF-5 was detectable in both cytoplasmic

    Article Snippet: In addition, in vivo binding of IRF-5 to relevant target gene promoters and their activation was reduced in the presence of vIRF-3 ( ).

    Techniques:

    A double α-helix motif in vIRF-3 is sufficient for binding of vIRF-3 to IRF-5. A , V5-tagged vIRF-3 constructs were expressed in HEK 293T cells and precipitated with anti-V5-loaded protein G-Sepharose beads. Cells transfected with empty V5-expression

    Journal:

    Article Title: The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 *The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 * S⃞

    doi: 10.1074/jbc.M809252200

    Figure Lengend Snippet: A double α-helix motif in vIRF-3 is sufficient for binding of vIRF-3 to IRF-5. A , V5-tagged vIRF-3 constructs were expressed in HEK 293T cells and precipitated with anti-V5-loaded protein G-Sepharose beads. Cells transfected with empty V5-expression

    Article Snippet: In addition, in vivo binding of IRF-5 to relevant target gene promoters and their activation was reduced in the presence of vIRF-3 ( ).

    Techniques: Binding Assay, Construct, Transfection, Expressing

    The double α-helix motif in vIRF-3 is necessary and sufficient for repression of the IFNβ promoter. Transient expression experiments were performed in HEK 293T cells. A reporter plasmid expressing luciferase under the control of the

    Journal:

    Article Title: The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 *The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 * S⃞

    doi: 10.1074/jbc.M809252200

    Figure Lengend Snippet: The double α-helix motif in vIRF-3 is necessary and sufficient for repression of the IFNβ promoter. Transient expression experiments were performed in HEK 293T cells. A reporter plasmid expressing luciferase under the control of the

    Article Snippet: In addition, in vivo binding of IRF-5 to relevant target gene promoters and their activation was reduced in the presence of vIRF-3 ( ).

    Techniques: Expressing, Plasmid Preparation, Luciferase

    Reduction of IRF-5-mediated ISRE reporter activity by vIRF-3 expression. HEK 293T cells were transfected with an ISRE-luciferase reporter construct and the indicated amounts of expression plasmids for IRF-5 or vIRF-3. Cells were co-transfected with

    Journal:

    Article Title: The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 *The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 * S⃞

    doi: 10.1074/jbc.M809252200

    Figure Lengend Snippet: Reduction of IRF-5-mediated ISRE reporter activity by vIRF-3 expression. HEK 293T cells were transfected with an ISRE-luciferase reporter construct and the indicated amounts of expression plasmids for IRF-5 or vIRF-3. Cells were co-transfected with

    Article Snippet: In addition, in vivo binding of IRF-5 to relevant target gene promoters and their activation was reduced in the presence of vIRF-3 ( ).

    Techniques: Activity Assay, Expressing, Transfection, Luciferase, Construct

    Inhibition of IRF-5-induced transcriptional transactivation by vIRF-3. HEK 293T cells were transfected with an ISRE-luciferase reporter construct and the indicated amounts of IRF-5, vIRF-3, and K3 expression constructs. Luciferase assays were performed

    Journal:

    Article Title: The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 *The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 * S⃞

    doi: 10.1074/jbc.M809252200

    Figure Lengend Snippet: Inhibition of IRF-5-induced transcriptional transactivation by vIRF-3. HEK 293T cells were transfected with an ISRE-luciferase reporter construct and the indicated amounts of IRF-5, vIRF-3, and K3 expression constructs. Luciferase assays were performed

    Article Snippet: In addition, in vivo binding of IRF-5 to relevant target gene promoters and their activation was reduced in the presence of vIRF-3 ( ).

    Techniques: Inhibition, Transfection, Luciferase, Construct, Expressing

    Interaction of vIRF-3 and IRF-5. A and B, in vitro co-immunoprecipitation of vIRF-3 and IRF-5. 293T cells were transfected with expression plasmids for one of three Myc-tagged proteins IRF-5, vIRF-3, or transglutaminase ( TGM ), respectively. Transfected

    Journal:

    Article Title: The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 *The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 * S⃞

    doi: 10.1074/jbc.M809252200

    Figure Lengend Snippet: Interaction of vIRF-3 and IRF-5. A and B, in vitro co-immunoprecipitation of vIRF-3 and IRF-5. 293T cells were transfected with expression plasmids for one of three Myc-tagged proteins IRF-5, vIRF-3, or transglutaminase ( TGM ), respectively. Transfected

    Article Snippet: In addition, in vivo binding of IRF-5 to relevant target gene promoters and their activation was reduced in the presence of vIRF-3 ( ).

    Techniques: In Vitro, Immunoprecipitation, Transfection, Expressing

    Knockdown of vIRF-3 renders PEL cells more sensitive to interferon treatment. Cells were either mock-transfected ( mock ; dark gray ), transfected with nonsense siRNA ( siN ; light gray ), or siRNA against vIRF-3 ( sivIRF-3 ; white). Eleven independent experiments

    Journal:

    Article Title: The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 *The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 * S⃞

    doi: 10.1074/jbc.M809252200

    Figure Lengend Snippet: Knockdown of vIRF-3 renders PEL cells more sensitive to interferon treatment. Cells were either mock-transfected ( mock ; dark gray ), transfected with nonsense siRNA ( siN ; light gray ), or siRNA against vIRF-3 ( sivIRF-3 ; white). Eleven independent experiments

    Article Snippet: In addition, in vivo binding of IRF-5 to relevant target gene promoters and their activation was reduced in the presence of vIRF-3 ( ).

    Techniques: Transfection

    Inhibition of IRF-5 DNA binding by vIRF-3 in PEL cells. Extracts from BC-3 cells treated with either nonsense or vIRF-3 siRNA were used for ISRE oligonucleotide pull-down analyses. Knockdown of vIRF-3 in PEL cells resulted in augmented binding of ISRE

    Journal:

    Article Title: The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 *The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 * S⃞

    doi: 10.1074/jbc.M809252200

    Figure Lengend Snippet: Inhibition of IRF-5 DNA binding by vIRF-3 in PEL cells. Extracts from BC-3 cells treated with either nonsense or vIRF-3 siRNA were used for ISRE oligonucleotide pull-down analyses. Knockdown of vIRF-3 in PEL cells resulted in augmented binding of ISRE

    Article Snippet: In addition, in vivo binding of IRF-5 to relevant target gene promoters and their activation was reduced in the presence of vIRF-3 ( ).

    Techniques: Inhibition, Binding Assay

    Influence of vIRF-3 on IRF-5 target gene expression. A , regulation of IRF-5 targets in transfected cells. Chromatin immunoprecipitation was carried out with cells transfected with either vector only, IRF-5-Myc expression plasmid, or both IRF-5-Myc

    Journal:

    Article Title: The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 *The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 * S⃞

    doi: 10.1074/jbc.M809252200

    Figure Lengend Snippet: Influence of vIRF-3 on IRF-5 target gene expression. A , regulation of IRF-5 targets in transfected cells. Chromatin immunoprecipitation was carried out with cells transfected with either vector only, IRF-5-Myc expression plasmid, or both IRF-5-Myc

    Article Snippet: In addition, in vivo binding of IRF-5 to relevant target gene promoters and their activation was reduced in the presence of vIRF-3 ( ).

    Techniques: Expressing, Transfection, Chromatin Immunoprecipitation, Plasmid Preparation

    Inhibition of IRF-5-mediated activation of the IFNβ promoter by vIRF-3. HEK 293T cells were transfected with an IFNβ promoter luciferase construct and the indicated amounts of IRF-5, vIRF-3, and LANA expression constructs. Bars represent

    Journal:

    Article Title: The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 *The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5 * S⃞

    doi: 10.1074/jbc.M809252200

    Figure Lengend Snippet: Inhibition of IRF-5-mediated activation of the IFNβ promoter by vIRF-3. HEK 293T cells were transfected with an IFNβ promoter luciferase construct and the indicated amounts of IRF-5, vIRF-3, and LANA expression constructs. Bars represent

    Article Snippet: In addition, in vivo binding of IRF-5 to relevant target gene promoters and their activation was reduced in the presence of vIRF-3 ( ).

    Techniques: Inhibition, Activation Assay, Transfection, Luciferase, Construct, Expressing

    Effect of HQ on AKT activation in IRF3 pathway (A) RAW264.7 cells (1×10 6 cells/ml) pretreated with either HQ (50 µM) or vehicle for 30 min were incubated with LPS (1.0 µg/ml) for the indicated time. Phosphorylated and total levels of TBK1 and AKT in total cell lysates were determined by Western blot analysis. (B) RAW264.7 cells (1×10 6 cells/ml) transfected with HA-AKT (1.0 µg/ml) for 24 h were treated with the indicated doses of HQ for another 24 h, and phosphorylated and total levels of IRF-3 and HA in total cell lysates were determined by Western blot analysis. (C) RAW264.7 cells (1×10 6 cells/ml) were transfected with either HA-AKT (1.0 µg/ml) or HA-AKT C310A (1.0 µg/ml) for 48 h. Phosphorylated and total levels of IRF-3, AKT, and HA in total cell lysates were determined by Western blot analysis. (D) RAW264.7 cells (1×10 6 cells/ml) transfected with either HA-AKT1 (1.0 µg/ ml), HA-AKT2 (1.0 µg/ml), or HA-AKT3 (1.0 µg/ml) for 24 h were treated with either HQ (50 µM) or vehicle for another 24 h. Phosphorylated and total levels of IRF-3, AKT, and HA in the total cell lysates were determined by Western blot analysis. (E) HEK293 cells (1×10 6 cells/ml) cotransfected with TBK1 (1.0 µg/ml), IRF-3-Luc (1.0 µg/ml), β-gal (0.1 µg/ml), and HA-AKT1 (1.0 µg/ml), HA-AKT2 (1.0 µg/ml), HA-AKT3 (1.0 µg/ml), or HA-AKT KD (kinase domain deletion mutant) for 24 h were treated with either HQ (50 µM) or vehicle for another 24 h. Luciferase activity was measured with a luminometer. All luciferase reporter gene activities were normalized to β-galactosidase activity. * p

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Hydroquinone suppresses IFN-β expression by targeting AKT/IRF3 pathway

    doi: 10.4196/kjpp.2017.21.5.547

    Figure Lengend Snippet: Effect of HQ on AKT activation in IRF3 pathway (A) RAW264.7 cells (1×10 6 cells/ml) pretreated with either HQ (50 µM) or vehicle for 30 min were incubated with LPS (1.0 µg/ml) for the indicated time. Phosphorylated and total levels of TBK1 and AKT in total cell lysates were determined by Western blot analysis. (B) RAW264.7 cells (1×10 6 cells/ml) transfected with HA-AKT (1.0 µg/ml) for 24 h were treated with the indicated doses of HQ for another 24 h, and phosphorylated and total levels of IRF-3 and HA in total cell lysates were determined by Western blot analysis. (C) RAW264.7 cells (1×10 6 cells/ml) were transfected with either HA-AKT (1.0 µg/ml) or HA-AKT C310A (1.0 µg/ml) for 48 h. Phosphorylated and total levels of IRF-3, AKT, and HA in total cell lysates were determined by Western blot analysis. (D) RAW264.7 cells (1×10 6 cells/ml) transfected with either HA-AKT1 (1.0 µg/ ml), HA-AKT2 (1.0 µg/ml), or HA-AKT3 (1.0 µg/ml) for 24 h were treated with either HQ (50 µM) or vehicle for another 24 h. Phosphorylated and total levels of IRF-3, AKT, and HA in the total cell lysates were determined by Western blot analysis. (E) HEK293 cells (1×10 6 cells/ml) cotransfected with TBK1 (1.0 µg/ml), IRF-3-Luc (1.0 µg/ml), β-gal (0.1 µg/ml), and HA-AKT1 (1.0 µg/ml), HA-AKT2 (1.0 µg/ml), HA-AKT3 (1.0 µg/ml), or HA-AKT KD (kinase domain deletion mutant) for 24 h were treated with either HQ (50 µM) or vehicle for another 24 h. Luciferase activity was measured with a luminometer. All luciferase reporter gene activities were normalized to β-galactosidase activity. * p

    Article Snippet: Antibodies specific for phosphorylated and total forms of IRF-3, TBK-1, AKT, hemagglutinin (HA) tag, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Activation Assay, Incubation, Western Blot, Transfection, Mutagenesis, Luciferase, Activity Assay

    Proposed model for HQ-mediated suppression of IFN-β production by targeting AKT kinases in the IRF-3 signaling pathway during macrophage-mediated inflammatory responses Bold denotes a signaling molecule targeted by HQ in macrophages during inflammatory responses.

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Hydroquinone suppresses IFN-β expression by targeting AKT/IRF3 pathway

    doi: 10.4196/kjpp.2017.21.5.547

    Figure Lengend Snippet: Proposed model for HQ-mediated suppression of IFN-β production by targeting AKT kinases in the IRF-3 signaling pathway during macrophage-mediated inflammatory responses Bold denotes a signaling molecule targeted by HQ in macrophages during inflammatory responses.

    Article Snippet: Antibodies specific for phosphorylated and total forms of IRF-3, TBK-1, AKT, hemagglutinin (HA) tag, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques:

    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

    Inhibition of EZH2 prevents repression of OCL negative factors and inhibits OCL formation. BMM cells were cultured with MCSF for 3 days (d-3 to d0) to form OCLp before addition of RANKL/MCSF to generate OCL. (A) OCLp were cultured with RANKL/MCSF in the presence of the indicated concentrations of GSK126 for 5 days. TRAP+ OCL formed were analyzed by microscopy (representative images on left) and the counts for multinucleated (n≥3) TRAP+ OCL/well graphed (right). (B) GSK126 (10 μM) was added at the indicated time intervals during the processes of OCLp and OCL (d4) formation and representative images and counts for multinucleated (n≥3) TRAP+ OCL/well are shown. (C) OCLp (d0) or stimulated with RANKL/MCSF +/− GSK126 (10 μM; GSK) for the indicated times were analyzed for Ezh2 mRNA by qPCR. (D) OCLp (d0) or stimulated with RANKL/MCSF for the indicated times were analyzed for total cellular levels of EZH2, H3K27me3, total H3 and βactin by WB. (E) OCLp cultures (d0) or after stimulation with RANKL/MCSF +/− GSK126 (10 μM) for 1 day were analyzed for total cellular levels of EZH2, H3K27me3, H3K9ac, H3 and βactin by WB. (F) OCLp (d0) or after stimulation with RANKL/MCSF +/− GSK126 (10 μM) for 1 or 5 days were analyzed for the expression of OCL-specific genes NFATc1 , RANK , CatK , DC-STAMP by qPCR. (G) OCLp (d0) or after stimulation with RANKL/MCSF +/− GSK126 (10 μM) for 1 day were analyzed for the expression of OCL-negative regulators MafB , Irf8 , Arg1 , and Bcl6 by qPCR. WB analyses show representative of 3 independent experiments and error bars in mRNA experiments represent SEM for 4-17 biological replicates. Statistical analysis used one-way ANOVA with Dunnett’s multiple comparisons test comparing to no GSK126 control (B), 0 time (C), or with Tukay’s multiple comparisons test for all means (G), and two-way ANOVA with Sidak multiple comparisons for day and treatment (F). Note that in C, d1 and d5 were not found to be significantly different from time 0.

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: EZH2 supports osteoclast differentiation and bone resorption via epigenetic and cytoplasmic targets

    doi: 10.1002/jbmr.3863

    Figure Lengend Snippet: Inhibition of EZH2 prevents repression of OCL negative factors and inhibits OCL formation. BMM cells were cultured with MCSF for 3 days (d-3 to d0) to form OCLp before addition of RANKL/MCSF to generate OCL. (A) OCLp were cultured with RANKL/MCSF in the presence of the indicated concentrations of GSK126 for 5 days. TRAP+ OCL formed were analyzed by microscopy (representative images on left) and the counts for multinucleated (n≥3) TRAP+ OCL/well graphed (right). (B) GSK126 (10 μM) was added at the indicated time intervals during the processes of OCLp and OCL (d4) formation and representative images and counts for multinucleated (n≥3) TRAP+ OCL/well are shown. (C) OCLp (d0) or stimulated with RANKL/MCSF +/− GSK126 (10 μM; GSK) for the indicated times were analyzed for Ezh2 mRNA by qPCR. (D) OCLp (d0) or stimulated with RANKL/MCSF for the indicated times were analyzed for total cellular levels of EZH2, H3K27me3, total H3 and βactin by WB. (E) OCLp cultures (d0) or after stimulation with RANKL/MCSF +/− GSK126 (10 μM) for 1 day were analyzed for total cellular levels of EZH2, H3K27me3, H3K9ac, H3 and βactin by WB. (F) OCLp (d0) or after stimulation with RANKL/MCSF +/− GSK126 (10 μM) for 1 or 5 days were analyzed for the expression of OCL-specific genes NFATc1 , RANK , CatK , DC-STAMP by qPCR. (G) OCLp (d0) or after stimulation with RANKL/MCSF +/− GSK126 (10 μM) for 1 day were analyzed for the expression of OCL-negative regulators MafB , Irf8 , Arg1 , and Bcl6 by qPCR. WB analyses show representative of 3 independent experiments and error bars in mRNA experiments represent SEM for 4-17 biological replicates. Statistical analysis used one-way ANOVA with Dunnett’s multiple comparisons test comparing to no GSK126 control (B), 0 time (C), or with Tukay’s multiple comparisons test for all means (G), and two-way ANOVA with Sidak multiple comparisons for day and treatment (F). Note that in C, d1 and d5 were not found to be significantly different from time 0.

    Article Snippet: EZH2 directed H3K27me3-dependent chromatin repression of developmental genes is central to cellular differentiation programs. A recent report by Qiao et al. demonstrated the importance of H3K27me3-based heterochromatin silencing of a subset of anti-inflammatory and pro-osteoclastogenic genes during INF-γ-mediated macrophage activation. The role of epigenetic-based gene suppression by EZH2 in combination with DNMT3a has only been reported for the IRF8 gene in a human OCL differentiation system. ( , ) The realization that EZH2 is a multifunctional histone methyltransferase, subjected to various regulatory post-translational modifications with both nuclear and cytoplasmic functions,( ) prompted us to better define the role of EZH2 during early osteoclastogenesis and in mature bone resorbing cells.

    Techniques: Inhibition, Cell Culture, Microscopy, Real-time Polymerase Chain Reaction, Western Blot, Expressing

    EZH2 is required for OCL differentiation. BMM cells were infected with lentivirus (LV) during OCLp formation with MCSF. (A, B) OCLp infected with SCR or sh- Ezh2 (EZH2-KD) LV were cultured with RANK/MCSF for 1 day and analyzed for (A) Ezh2 and MafB expression by qPCR and (B) total cellular levels of EZH2, H3K27me3, MafB and βactin by WB. (C) SCR and EZH2-KD OCLp were stimulated with RANKL/MCSF for 4 days and analyzed for OCL formation. Representative images (left) and counts for multinucleated (n≥3) TRAP+ OCL/well are shown. (D, E) OCLp cells infected with control or EZH2-OE LV were stimulated with RANKL/MCSF for 1 day and analyzed for (D) Ezh2 and MafB expression by qPCR and (E) total cellular levels of EZH2, H3K27me3, H3 and βactin by WB. (F) Control and EZH2-OE OCLp were stimulated with RANKL/MCSF for 3 days and analyzed for OCL formation. Representative microscopy images (left) and counts for multinucleated (n≥3) TRAP+ OCL/well are shown. (G) Control and EZH2-OE OCLp were plated on bone slices in the presence of RANKL/MCSF for 8 days. Representative microscopy images for Toluidine blue stained resorption pits are shown and graphs (with SD) represent ImageJ quantitation of 5 distinct OCL resorption areas, counts for multinucleated (n≥3) TRAP+ OCL/bone area (μm 2 ), and resorption per OCL. WB, TRAP assays and resorption experiments were conducted at least 2 independent times. Error bars in mRNA experiments represent SEM for 2-5 biological replicates. Statistical analyses used Student’s t test.

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: EZH2 supports osteoclast differentiation and bone resorption via epigenetic and cytoplasmic targets

    doi: 10.1002/jbmr.3863

    Figure Lengend Snippet: EZH2 is required for OCL differentiation. BMM cells were infected with lentivirus (LV) during OCLp formation with MCSF. (A, B) OCLp infected with SCR or sh- Ezh2 (EZH2-KD) LV were cultured with RANK/MCSF for 1 day and analyzed for (A) Ezh2 and MafB expression by qPCR and (B) total cellular levels of EZH2, H3K27me3, MafB and βactin by WB. (C) SCR and EZH2-KD OCLp were stimulated with RANKL/MCSF for 4 days and analyzed for OCL formation. Representative images (left) and counts for multinucleated (n≥3) TRAP+ OCL/well are shown. (D, E) OCLp cells infected with control or EZH2-OE LV were stimulated with RANKL/MCSF for 1 day and analyzed for (D) Ezh2 and MafB expression by qPCR and (E) total cellular levels of EZH2, H3K27me3, H3 and βactin by WB. (F) Control and EZH2-OE OCLp were stimulated with RANKL/MCSF for 3 days and analyzed for OCL formation. Representative microscopy images (left) and counts for multinucleated (n≥3) TRAP+ OCL/well are shown. (G) Control and EZH2-OE OCLp were plated on bone slices in the presence of RANKL/MCSF for 8 days. Representative microscopy images for Toluidine blue stained resorption pits are shown and graphs (with SD) represent ImageJ quantitation of 5 distinct OCL resorption areas, counts for multinucleated (n≥3) TRAP+ OCL/bone area (μm 2 ), and resorption per OCL. WB, TRAP assays and resorption experiments were conducted at least 2 independent times. Error bars in mRNA experiments represent SEM for 2-5 biological replicates. Statistical analyses used Student’s t test.

    Article Snippet: EZH2 directed H3K27me3-dependent chromatin repression of developmental genes is central to cellular differentiation programs. A recent report by Qiao et al. demonstrated the importance of H3K27me3-based heterochromatin silencing of a subset of anti-inflammatory and pro-osteoclastogenic genes during INF-γ-mediated macrophage activation. The role of epigenetic-based gene suppression by EZH2 in combination with DNMT3a has only been reported for the IRF8 gene in a human OCL differentiation system. ( , ) The realization that EZH2 is a multifunctional histone methyltransferase, subjected to various regulatory post-translational modifications with both nuclear and cytoplasmic functions,( ) prompted us to better define the role of EZH2 during early osteoclastogenesis and in mature bone resorbing cells.

    Techniques: Infection, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Microscopy, Staining, Quantitation Assay

    Cytoplasmic EZH2 methyltransferase activity is required for OCL resorption. OCLp were plated onto bone slices, and stimulated with MCSF alone or RANKL/MCSF for 9 days, except the sample on the far right which was harvested at day 7 as a control. GSK126 (10 μM) was applied to cultures at day 0 or day 7 as indicated. The fixed cells were stained for TRAP and also fluorescently stained with Phalloidin rhodamine (red) and DAPI (blue). (A) The TRAP stained OCL (Brightfield microscopy top panels) are shown using widefield fluorescent microscopy together with the fluorescent stains Phalloidin (red in middle panels) and DAPI (blue in bottom panels). The dark areas in the fluorescent images reveal the presence of the TRAP stain through interference. Scale bars represent 20 μm. (B) The cells were removed and Toluidine blue was applied to visualize OCL resorbed areas. (C) ImageJ was used to quantitate the resorption area. (D) Counts of TRAP+ multinuclear OCL/bone area (μm 2 ) formed on the bone surface. Statistical analysis used two-way ANOVA with Tukay’s multiple comparisons test for all means.

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: EZH2 supports osteoclast differentiation and bone resorption via epigenetic and cytoplasmic targets

    doi: 10.1002/jbmr.3863

    Figure Lengend Snippet: Cytoplasmic EZH2 methyltransferase activity is required for OCL resorption. OCLp were plated onto bone slices, and stimulated with MCSF alone or RANKL/MCSF for 9 days, except the sample on the far right which was harvested at day 7 as a control. GSK126 (10 μM) was applied to cultures at day 0 or day 7 as indicated. The fixed cells were stained for TRAP and also fluorescently stained with Phalloidin rhodamine (red) and DAPI (blue). (A) The TRAP stained OCL (Brightfield microscopy top panels) are shown using widefield fluorescent microscopy together with the fluorescent stains Phalloidin (red in middle panels) and DAPI (blue in bottom panels). The dark areas in the fluorescent images reveal the presence of the TRAP stain through interference. Scale bars represent 20 μm. (B) The cells were removed and Toluidine blue was applied to visualize OCL resorbed areas. (C) ImageJ was used to quantitate the resorption area. (D) Counts of TRAP+ multinuclear OCL/bone area (μm 2 ) formed on the bone surface. Statistical analysis used two-way ANOVA with Tukay’s multiple comparisons test for all means.

    Article Snippet: EZH2 directed H3K27me3-dependent chromatin repression of developmental genes is central to cellular differentiation programs. A recent report by Qiao et al. demonstrated the importance of H3K27me3-based heterochromatin silencing of a subset of anti-inflammatory and pro-osteoclastogenic genes during INF-γ-mediated macrophage activation. The role of epigenetic-based gene suppression by EZH2 in combination with DNMT3a has only been reported for the IRF8 gene in a human OCL differentiation system. ( , ) The realization that EZH2 is a multifunctional histone methyltransferase, subjected to various regulatory post-translational modifications with both nuclear and cytoplasmic functions,( ) prompted us to better define the role of EZH2 during early osteoclastogenesis and in mature bone resorbing cells.

    Techniques: Activity Assay, Staining, Microscopy

    Summary for nuclear and cytoplasmic roles for EZH2 during osteoclastogenesis. EZH2 is a RANKL inducible positive regulator of OCL differentiation required for silencing of MafB expression by both adding H3K27me3 as well as increasing C/EBPβ-LIP at the MafB gene promoter. Therefore, blocking EZH2 methyltransferase activity with GSK126 de-represses MafB both by decreased epigenetic silencing and by lack of proper RANKL-activation of the PI3K-pAKT-pmTOR signaling pathway. This results in increased C/EBPβ-LAP isoform levels with its enhanced binding/activation of the MafB gene. Since blockade of EZH2 methyltransferase activity in mature OCL results in condensed cytoskeletal architecture, impaired F-actin ring formation and reduced OCL resorption, we hypothesize that by modulating actin dynamics, EZH2 modulates cytoskeletal rearrangements in mature OCL.

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: EZH2 supports osteoclast differentiation and bone resorption via epigenetic and cytoplasmic targets

    doi: 10.1002/jbmr.3863

    Figure Lengend Snippet: Summary for nuclear and cytoplasmic roles for EZH2 during osteoclastogenesis. EZH2 is a RANKL inducible positive regulator of OCL differentiation required for silencing of MafB expression by both adding H3K27me3 as well as increasing C/EBPβ-LIP at the MafB gene promoter. Therefore, blocking EZH2 methyltransferase activity with GSK126 de-represses MafB both by decreased epigenetic silencing and by lack of proper RANKL-activation of the PI3K-pAKT-pmTOR signaling pathway. This results in increased C/EBPβ-LAP isoform levels with its enhanced binding/activation of the MafB gene. Since blockade of EZH2 methyltransferase activity in mature OCL results in condensed cytoskeletal architecture, impaired F-actin ring formation and reduced OCL resorption, we hypothesize that by modulating actin dynamics, EZH2 modulates cytoskeletal rearrangements in mature OCL.

    Article Snippet: EZH2 directed H3K27me3-dependent chromatin repression of developmental genes is central to cellular differentiation programs. A recent report by Qiao et al. demonstrated the importance of H3K27me3-based heterochromatin silencing of a subset of anti-inflammatory and pro-osteoclastogenic genes during INF-γ-mediated macrophage activation. The role of epigenetic-based gene suppression by EZH2 in combination with DNMT3a has only been reported for the IRF8 gene in a human OCL differentiation system. ( , ) The realization that EZH2 is a multifunctional histone methyltransferase, subjected to various regulatory post-translational modifications with both nuclear and cytoplasmic functions,( ) prompted us to better define the role of EZH2 during early osteoclastogenesis and in mature bone resorbing cells.

    Techniques: Expressing, Blocking Assay, Activity Assay, Activation Assay, Binding Assay

    GSK126 treatment of mature OCL impairs cytoskeletal architecture and F-actin ring formation. OCLp were plated onto bone slices and either maintained in MCSF or cultured with RANKL/MCSF for 8 days to generate mature OCL as indicated. (A) GSK126 (10 μM) was added to the OCL at day 6 (bottom panels) and the fixed cells were immunofluorescently stained for CTR (red), EZH2 (green), and DAPI (blue). (B) OCL were formed by RANKL/MCSF alone or with GSK126 (10 μM) added for the entire time (starting at d0) or only added to the OCL at day 6 as indicated. The fixed cells were fluorescently stained with Phalloidin rhodamine to detect actin (red) and DAPI (blue). All images represent fluorescent microscopy confocal sections and scale bars indicate 20 μm.

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: EZH2 supports osteoclast differentiation and bone resorption via epigenetic and cytoplasmic targets

    doi: 10.1002/jbmr.3863

    Figure Lengend Snippet: GSK126 treatment of mature OCL impairs cytoskeletal architecture and F-actin ring formation. OCLp were plated onto bone slices and either maintained in MCSF or cultured with RANKL/MCSF for 8 days to generate mature OCL as indicated. (A) GSK126 (10 μM) was added to the OCL at day 6 (bottom panels) and the fixed cells were immunofluorescently stained for CTR (red), EZH2 (green), and DAPI (blue). (B) OCL were formed by RANKL/MCSF alone or with GSK126 (10 μM) added for the entire time (starting at d0) or only added to the OCL at day 6 as indicated. The fixed cells were fluorescently stained with Phalloidin rhodamine to detect actin (red) and DAPI (blue). All images represent fluorescent microscopy confocal sections and scale bars indicate 20 μm.

    Article Snippet: EZH2 directed H3K27me3-dependent chromatin repression of developmental genes is central to cellular differentiation programs. A recent report by Qiao et al. demonstrated the importance of H3K27me3-based heterochromatin silencing of a subset of anti-inflammatory and pro-osteoclastogenic genes during INF-γ-mediated macrophage activation. The role of epigenetic-based gene suppression by EZH2 in combination with DNMT3a has only been reported for the IRF8 gene in a human OCL differentiation system. ( , ) The realization that EZH2 is a multifunctional histone methyltransferase, subjected to various regulatory post-translational modifications with both nuclear and cytoplasmic functions,( ) prompted us to better define the role of EZH2 during early osteoclastogenesis and in mature bone resorbing cells.

    Techniques: Cell Culture, Staining, Microscopy

    Src family kinases are required for interferon regulatory factor 1 (IRF-1) protein accumulation in response to TLR7/8 stimulation in monocytes and B cells. (A,C) Immunoblot analysis of IRF-1 and p-SFKs in THP-1 (A) and primary B cells (C) pretreated with PP2 (20 µM) or DMSO alone and stimulated with R848 (10 µM) for 2 h. After stripping, filters were re-probed with an antibody against actin as loading control. Results are representative at least of three independent experiments. The histograms on the right of the blot show the results of the densitometric analysis on three independent experiments. Each signal was normalized to its respective actin signal on the same blot and expressed as fold induction (mean ± SD) compared to untreated sample. (B,D) qRT-PCR analysis of lRF-1 mRNA in THP-1 (B) and human B cells (D) pretreated with PP2 (20 µM) or DMSO alone and stimulated with R848 (10 µM) for 2 h. The relative abundance of the gene transcripts was determined on triplicate samples from at least three independent experiments using the ΔΔCt method and is expressed as the normalized fold expression (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: Src family kinases are required for interferon regulatory factor 1 (IRF-1) protein accumulation in response to TLR7/8 stimulation in monocytes and B cells. (A,C) Immunoblot analysis of IRF-1 and p-SFKs in THP-1 (A) and primary B cells (C) pretreated with PP2 (20 µM) or DMSO alone and stimulated with R848 (10 µM) for 2 h. After stripping, filters were re-probed with an antibody against actin as loading control. Results are representative at least of three independent experiments. The histograms on the right of the blot show the results of the densitometric analysis on three independent experiments. Each signal was normalized to its respective actin signal on the same blot and expressed as fold induction (mean ± SD) compared to untreated sample. (B,D) qRT-PCR analysis of lRF-1 mRNA in THP-1 (B) and human B cells (D) pretreated with PP2 (20 µM) or DMSO alone and stimulated with R848 (10 µM) for 2 h. The relative abundance of the gene transcripts was determined on triplicate samples from at least three independent experiments using the ΔΔCt method and is expressed as the normalized fold expression (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: Stripping Membranes, Quantitative RT-PCR, Expressing

    Src family kinases activation is essential for interferon regulatory factor 1 (IRF-1) K63-linked ubiquitination. (A) Immunoblot analysis of IRF-1 and pSFKs in hTLR7-HEK293 cells pretreated or not with PP2 (20 µM) for 30 min and stimulated with R848 (10 µM) for 2 h. After stripping, membranes were blotted with anti-actin antibody as loading control. The histograms on the right of the blots show the results of the densitometric analysis on three independent experiments. Each signal was normalized to its respective actin signal on the same blot and expressed as fold induction (mean ± SD) compared to untreated sample. (B,C) hTLR7-HEK293 cells transiently transfected with plasmids coding for hemagglutinin (HA)-tagged K63-only (B) or K48-only ubiquitin mutants (C) 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-IRF-1 antibody, separated by SDS-PAGE, and immunoblotted using anti-HA antibody for ubiquitin detection. Membranes were stripped and re-probed with an anti-IRF-1 antibody as control (bottom panels). The histograms on the right of the blots show the results of the densitometric analysis 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: Src family kinases activation is essential for interferon regulatory factor 1 (IRF-1) K63-linked ubiquitination. (A) Immunoblot analysis of IRF-1 and pSFKs in hTLR7-HEK293 cells pretreated or not with PP2 (20 µM) for 30 min and stimulated with R848 (10 µM) for 2 h. After stripping, membranes were blotted with anti-actin antibody as loading control. The histograms on the right of the blots show the results of the densitometric analysis on three independent experiments. Each signal was normalized to its respective actin signal on the same blot and expressed as fold induction (mean ± SD) compared to untreated sample. (B,C) hTLR7-HEK293 cells transiently transfected with plasmids coding for hemagglutinin (HA)-tagged K63-only (B) or K48-only ubiquitin mutants (C) 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-IRF-1 antibody, separated by SDS-PAGE, and immunoblotted using anti-HA antibody for ubiquitin detection. Membranes were stripped and re-probed with an anti-IRF-1 antibody as control (bottom panels). The histograms on the right of the blots show the results of the densitometric analysis 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: Activation Assay, Stripping Membranes, Transfection, Immunoprecipitation, SDS Page

    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

    BET inhibitors do not suppress IFN-γ-induced IRF1 protein expression. ( A ) Primary PSCs and the pancreatic stellate cell line were pre-treated with the BET inhibitors JQ1 (1 μM) or I-BET151 (I-BET; 1 μM) for 30 minutes and then treated with IFN-γ (0.2 μg/mL) for 4 hours. The effect on IRF1 mRNA and protein expression was determined by qRT-PCR and by Western blotting. ( B ) Primary PSCs and the pancreatic stellate cell line were transfected with control siRNA or with siRNA against BRD4 for 48 hours. The cells were then treated with IFN-γ (0.2 μg/mL) for 4 hours. The effect on BRD4 protein was determined by qRT-PCR and by Western blotting, and the effect on IRF1 mRNA was determined by qRT-PCR. Bands from three independent experiments were quantified by densitometry and data expressed as relative ratios. The gene expression results are representative of three independent experiments. Bar graphs represent means +/− S.D. ns, not significant; *p

    Journal: Scientific Reports

    Article Title: Interplay between interferon regulatory factor 1 and BRD4 in the regulation of PD-L1 in pancreatic stellate cells

    doi: 10.1038/s41598-018-31658-1

    Figure Lengend Snippet: BET inhibitors do not suppress IFN-γ-induced IRF1 protein expression. ( A ) Primary PSCs and the pancreatic stellate cell line were pre-treated with the BET inhibitors JQ1 (1 μM) or I-BET151 (I-BET; 1 μM) for 30 minutes and then treated with IFN-γ (0.2 μg/mL) for 4 hours. The effect on IRF1 mRNA and protein expression was determined by qRT-PCR and by Western blotting. ( B ) Primary PSCs and the pancreatic stellate cell line were transfected with control siRNA or with siRNA against BRD4 for 48 hours. The cells were then treated with IFN-γ (0.2 μg/mL) for 4 hours. The effect on BRD4 protein was determined by qRT-PCR and by Western blotting, and the effect on IRF1 mRNA was determined by qRT-PCR. Bands from three independent experiments were quantified by densitometry and data expressed as relative ratios. The gene expression results are representative of three independent experiments. Bar graphs represent means +/− S.D. ns, not significant; *p

    Article Snippet: Antibodies against c-MYC (5605), human PD-L1 (E1L3N clone; 13684), IRF1 (8478) and control IgG (2729) antibody for ChIP were purchased from Cell Signaling Technology.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection

    BET inhibitors decrease binding of IRF1 to PD-L1 promoter. ( A , B ) The pancreatic stellate cell line was pre-treated with the BET inhibitor JQ1 (1 μM) for 30 minutes and then treated with IFN-γ (0.2 μg/mL) for 4 hours. The cells were subjected to ChIP analysis using anti-BRD4 antibody ( A ) or anti-IRF1 antibody ( B ). An isotype-matched IgG was used as a negative control. The association with the PD-L1 gene promoter was quantified by qPCR. The results are representative of three independent experiments. Bar graphs represent means +/−S.D. **p

    Journal: Scientific Reports

    Article Title: Interplay between interferon regulatory factor 1 and BRD4 in the regulation of PD-L1 in pancreatic stellate cells

    doi: 10.1038/s41598-018-31658-1

    Figure Lengend Snippet: BET inhibitors decrease binding of IRF1 to PD-L1 promoter. ( A , B ) The pancreatic stellate cell line was pre-treated with the BET inhibitor JQ1 (1 μM) for 30 minutes and then treated with IFN-γ (0.2 μg/mL) for 4 hours. The cells were subjected to ChIP analysis using anti-BRD4 antibody ( A ) or anti-IRF1 antibody ( B ). An isotype-matched IgG was used as a negative control. The association with the PD-L1 gene promoter was quantified by qPCR. The results are representative of three independent experiments. Bar graphs represent means +/−S.D. **p

    Article Snippet: Antibodies against c-MYC (5605), human PD-L1 (E1L3N clone; 13684), IRF1 (8478) and control IgG (2729) antibody for ChIP were purchased from Cell Signaling Technology.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction

    IRF1 knockdown decreases IFN-γ-induced PD-L1 expression. ( A , B ) Primary PSCs and the pancreatic stellate cell line were transfected with control siRNA or with siRNA against IRF1 for 48 hours. The cells were then treated with IFN-γ (0.2 μg/mL) for 24 hours. The effect on IRF1 was determined by qRT-PCR and by Western blotting, and the effect on PD-L1 mRNA was determined by qRT-PCR. IRF1 and GAPDH bands from three independent experiments were quantified by densitometry and data expressed as relative ratios. The gene expression results are representative of three independent experiments. Bar graphs represent means +/−S.D. ns, not significant; *p

    Journal: Scientific Reports

    Article Title: Interplay between interferon regulatory factor 1 and BRD4 in the regulation of PD-L1 in pancreatic stellate cells

    doi: 10.1038/s41598-018-31658-1

    Figure Lengend Snippet: IRF1 knockdown decreases IFN-γ-induced PD-L1 expression. ( A , B ) Primary PSCs and the pancreatic stellate cell line were transfected with control siRNA or with siRNA against IRF1 for 48 hours. The cells were then treated with IFN-γ (0.2 μg/mL) for 24 hours. The effect on IRF1 was determined by qRT-PCR and by Western blotting, and the effect on PD-L1 mRNA was determined by qRT-PCR. IRF1 and GAPDH bands from three independent experiments were quantified by densitometry and data expressed as relative ratios. The gene expression results are representative of three independent experiments. Bar graphs represent means +/−S.D. ns, not significant; *p

    Article Snippet: Antibodies against c-MYC (5605), human PD-L1 (E1L3N clone; 13684), IRF1 (8478) and control IgG (2729) antibody for ChIP were purchased from Cell Signaling Technology.

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot

    Regulation of HMX2 and HMX3 by KMT2A and ERK-signalling. (A) RQ-PCR analysis of EOL-1 (left) and MV4-11 (middle) treated for siRNA-mediated knockdown of KMT2A demonstrates raised expression levels of HMX2 and HMX3, indicating a repressive impact of KMT2A. This treatment resulted in enhanced expression of HMX3 in HL-60 cells which did not express HMX2 (right). Asterisks indicate calculated p-values obtained by t-Test analysis of controls (siCTR) and siRNA-targeted knockdown. (B) RQ-PCR analysis of KMT2A of EOL-1 cells treated for siRNA-mediated knockdown of HMX2 (left) and HMX3 (right) showed unaltered KMT2A expression levels, discounting any regulatory impact. (C) Quantification of KMT2A transcripts in AML cell lines and primary granulocytes showed low expression levels in EOL-1, MV4-11 and in eosinophils. (D) RQ-PCR analysis of HMX2 showed elevated expression levels in EOL-1 cells treated with PDGFRA-inhibitor dasatinib. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (DMSO) and pharmacological treatments. (E) Treatment of EOL-1 cells with ERK-inhbitor PD98059 resulted in reduced levels of phospho-ERK as analyzed by Western blot (left), and in elevated transcript levels of HMX2 (middle) and HMX3 (right) as analyzed by RQ-PCR. (F) Treatment of EOL-1 cells with ERK-activator FLT3LG resulted in elevated levels of phosphorylated ERK (above) and in reduced transcript levels of HMX2 and HMX3 (below).

    Journal: PLoS ONE

    Article Title: Aberrant expression of NKL homeobox genes HMX2 and HMX3 interferes with cell differentiation in acute myeloid leukemia

    doi: 10.1371/journal.pone.0240120

    Figure Lengend Snippet: Regulation of HMX2 and HMX3 by KMT2A and ERK-signalling. (A) RQ-PCR analysis of EOL-1 (left) and MV4-11 (middle) treated for siRNA-mediated knockdown of KMT2A demonstrates raised expression levels of HMX2 and HMX3, indicating a repressive impact of KMT2A. This treatment resulted in enhanced expression of HMX3 in HL-60 cells which did not express HMX2 (right). Asterisks indicate calculated p-values obtained by t-Test analysis of controls (siCTR) and siRNA-targeted knockdown. (B) RQ-PCR analysis of KMT2A of EOL-1 cells treated for siRNA-mediated knockdown of HMX2 (left) and HMX3 (right) showed unaltered KMT2A expression levels, discounting any regulatory impact. (C) Quantification of KMT2A transcripts in AML cell lines and primary granulocytes showed low expression levels in EOL-1, MV4-11 and in eosinophils. (D) RQ-PCR analysis of HMX2 showed elevated expression levels in EOL-1 cells treated with PDGFRA-inhibitor dasatinib. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (DMSO) and pharmacological treatments. (E) Treatment of EOL-1 cells with ERK-inhbitor PD98059 resulted in reduced levels of phospho-ERK as analyzed by Western blot (left), and in elevated transcript levels of HMX2 (middle) and HMX3 (right) as analyzed by RQ-PCR. (F) Treatment of EOL-1 cells with ERK-activator FLT3LG resulted in elevated levels of phosphorylated ERK (above) and in reduced transcript levels of HMX2 and HMX3 (below).

    Article Snippet: The results confirmed mutual activation which was more prominent for HMX3 in MV4-11.

    Techniques: Polymerase Chain Reaction, Expressing, Western Blot

    Regulation of EPX by HMX2 and HMX3. (A) Quantification of EPX expression in primary granulocytes (neutrophils, eosinophils, basophils) and selected cell lines by RQ-PCR. (B) SiRNA-mediated knockdown of HMX2 (left) and HMX3 (right) resulted in elevated expression levels of EPX. Reduced expression of HMX2 and HMX3 and elevated expression of EPX was also shown by Western blot analysis. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (siCTR) and siRNA-targeted knockdown. (C) Forced expression of HMX2 in HL-60 cells resulted in reduced expression level of EPX (left). SiRNA-mediated knockdown of EPX resulted in activation of CD11B expression (right). (D) A genomic map of the locus for EPX was obtained from the UCSC genome browser, showing potential transcription factor binding sites including one for HMX1. (E) Reporter-gene assay in NIH-3T3 cells using a fragment containing the identified HMX-site. Forced expression of HMX2 resulted in decreased reporter-gene activity, demonstrating a direct repressive impact. (F) Forced expression of EPX in EOL-1 slightly reduced the expression level of CD11B. Microscopical inspection of Giemsa-May-Grünwald-stained EOL-1 cells indicated strongly induced apoptosis by forced EPX expression as possible consequence of continued differentiation. Living cells resemble differentiated cells after DMSO-induction.

    Journal: PLoS ONE

    Article Title: Aberrant expression of NKL homeobox genes HMX2 and HMX3 interferes with cell differentiation in acute myeloid leukemia

    doi: 10.1371/journal.pone.0240120

    Figure Lengend Snippet: Regulation of EPX by HMX2 and HMX3. (A) Quantification of EPX expression in primary granulocytes (neutrophils, eosinophils, basophils) and selected cell lines by RQ-PCR. (B) SiRNA-mediated knockdown of HMX2 (left) and HMX3 (right) resulted in elevated expression levels of EPX. Reduced expression of HMX2 and HMX3 and elevated expression of EPX was also shown by Western blot analysis. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (siCTR) and siRNA-targeted knockdown. (C) Forced expression of HMX2 in HL-60 cells resulted in reduced expression level of EPX (left). SiRNA-mediated knockdown of EPX resulted in activation of CD11B expression (right). (D) A genomic map of the locus for EPX was obtained from the UCSC genome browser, showing potential transcription factor binding sites including one for HMX1. (E) Reporter-gene assay in NIH-3T3 cells using a fragment containing the identified HMX-site. Forced expression of HMX2 resulted in decreased reporter-gene activity, demonstrating a direct repressive impact. (F) Forced expression of EPX in EOL-1 slightly reduced the expression level of CD11B. Microscopical inspection of Giemsa-May-Grünwald-stained EOL-1 cells indicated strongly induced apoptosis by forced EPX expression as possible consequence of continued differentiation. Living cells resemble differentiated cells after DMSO-induction.

    Article Snippet: The results confirmed mutual activation which was more prominent for HMX3 in MV4-11.

    Techniques: Expressing, Polymerase Chain Reaction, Western Blot, Activation Assay, Binding Assay, Reporter Gene Assay, Activity Assay, Staining

    Gene regulatory network for HMX2 and HMX3 in AML. This diagram summarizes the results of this study, showing a gene regulatory network. HMX2 and HMX3 are located centrally, activating and repressing factors and pathways are indicated above, and the target genes EPX and HTR7 below. Deregulation of EPX and HTR7 indicates aberrant effects for differentiation and proliferation. Genomic rearrangements targeting KMT2A or PDGFRA are indicated.

    Journal: PLoS ONE

    Article Title: Aberrant expression of NKL homeobox genes HMX2 and HMX3 interferes with cell differentiation in acute myeloid leukemia

    doi: 10.1371/journal.pone.0240120

    Figure Lengend Snippet: Gene regulatory network for HMX2 and HMX3 in AML. This diagram summarizes the results of this study, showing a gene regulatory network. HMX2 and HMX3 are located centrally, activating and repressing factors and pathways are indicated above, and the target genes EPX and HTR7 below. Deregulation of EPX and HTR7 indicates aberrant effects for differentiation and proliferation. Genomic rearrangements targeting KMT2A or PDGFRA are indicated.

    Article Snippet: The results confirmed mutual activation which was more prominent for HMX3 in MV4-11.

    Techniques:

    Differentiation of EOL-1. (A) Treatment of EOL-1 cells with DMSO for three days induced morphological alterations as shown by Giemsa-May-Grünwald staining. The nuclei became typically indented like kidney beans which can be interpreted as eosinophilic differentiation. (B) The same treatment resulted in decreased expression of HMX2 (left) and HMX3 (right) as analyzed by RQ-PCR. SiRNA-mediated knockdown of HMX2 (C) or treatment with TNFa (D) generated the same nuclear indentation as visible after DMSO-treatment, indicating that HMX2/3 inhibited eosinophilic differentiation. (E) Marker expression. (F) Marker expression. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (siCTR) and siRNA-targeted knockdown.

    Journal: PLoS ONE

    Article Title: Aberrant expression of NKL homeobox genes HMX2 and HMX3 interferes with cell differentiation in acute myeloid leukemia

    doi: 10.1371/journal.pone.0240120

    Figure Lengend Snippet: Differentiation of EOL-1. (A) Treatment of EOL-1 cells with DMSO for three days induced morphological alterations as shown by Giemsa-May-Grünwald staining. The nuclei became typically indented like kidney beans which can be interpreted as eosinophilic differentiation. (B) The same treatment resulted in decreased expression of HMX2 (left) and HMX3 (right) as analyzed by RQ-PCR. SiRNA-mediated knockdown of HMX2 (C) or treatment with TNFa (D) generated the same nuclear indentation as visible after DMSO-treatment, indicating that HMX2/3 inhibited eosinophilic differentiation. (E) Marker expression. (F) Marker expression. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (siCTR) and siRNA-targeted knockdown.

    Article Snippet: The results confirmed mutual activation which was more prominent for HMX3 in MV4-11.

    Techniques: Staining, Expressing, Polymerase Chain Reaction, Generated, Marker

    Expression of HMX genes in AML cell lines. (A) Gene expression values (DESeq2 normalized count data) are shown according to RNA-seq data from the LL-100 dataset for HMX2 (above) and HMX3 (below). The color-code indicates AML-subgroups: erythroid (brown), megakaryocytic (yellow), monocytic (orange), myelocytic (violet); CML-subtypes: myeloid (green), lymphoid (blue), myeloproliferativ neoplasm (just cell line SET-2). Of note, the scale illustrating the transcript amount is much smaller for HMX1. According to the setting of the cut-off at 500 normalized counts no cell line is positive for HMX1. The expression of HMX2 (B) and HMX3 (C) was analyzed in selected cell lines and primary samples from granulocytes by RQ-PCR and Western blot (inserts). TUBA served as loading control. Significant expression was detected in cell lines EOL-1, MOLM-13 and MV4-11 which all carry KMT2A aberrations. Of note, HL-60 expressed low levels of HMX3 but no HMX2 RNA. WT: wild type.

    Journal: PLoS ONE

    Article Title: Aberrant expression of NKL homeobox genes HMX2 and HMX3 interferes with cell differentiation in acute myeloid leukemia

    doi: 10.1371/journal.pone.0240120

    Figure Lengend Snippet: Expression of HMX genes in AML cell lines. (A) Gene expression values (DESeq2 normalized count data) are shown according to RNA-seq data from the LL-100 dataset for HMX2 (above) and HMX3 (below). The color-code indicates AML-subgroups: erythroid (brown), megakaryocytic (yellow), monocytic (orange), myelocytic (violet); CML-subtypes: myeloid (green), lymphoid (blue), myeloproliferativ neoplasm (just cell line SET-2). Of note, the scale illustrating the transcript amount is much smaller for HMX1. According to the setting of the cut-off at 500 normalized counts no cell line is positive for HMX1. The expression of HMX2 (B) and HMX3 (C) was analyzed in selected cell lines and primary samples from granulocytes by RQ-PCR and Western blot (inserts). TUBA served as loading control. Significant expression was detected in cell lines EOL-1, MOLM-13 and MV4-11 which all carry KMT2A aberrations. Of note, HL-60 expressed low levels of HMX3 but no HMX2 RNA. WT: wild type.

    Article Snippet: The results confirmed mutual activation which was more prominent for HMX3 in MV4-11.

    Techniques: Expressing, RNA Sequencing Assay, Polymerase Chain Reaction, Western Blot

    Identification of upstream factors regulating HMX2 and HMX3. (A) Treatment of EOL-1 cells with IL7 resulted in increased transcript levels of HMX2 and HMX3 as analyzed by RQ-PCR. Asterisks indicate calculated p-values obtained by t-Test analysis of medium controls and interleukin stimulation. (B) Treatment of EOL-1 and MV4-11 cells with WNT3A and WNT5B resulted in increased expression levels of HMX2 (left) and HMX3 (right). (C) Treatment of EOL-1 cells with NFkB-activator TNFa resulted in reduced expression levels of HMX2 and HMX3 (left). Treatment of EOL-1 cells with NFkB-inhibitor resulted in increased expression of HMX2 and HMX3 (middle). STAT5A served as marker for NFkB-activity which showed low expression levels and increased activity after stimulation with TNFa (right). (D) A genomic map of the locus for HMX2/3 was obtained from the UCSC genome browser, showing potential transcription factor binding sites including those for IRF1, IRF2 and NKX2-5. (E) RQ-PCR analysis of EOL-1 cells treated for siRNA-mediated knockdown of IRF8 resulted in elevated expression levels of HMX2 (left) and HMX3 (right), indicating an activating impact of IRF8. (F) Quantification of IRF8 by RQ-PCR in primary granulocytes and monocytes (left) and selected AML cell lines (right) demonstrated significant levels in EOL-1 and MV4-11. (G) RQ-PCR analysis of EOL-1 (left) and MV4-11 (right) treated for siRNA-mediated knockdown of HMX3 and HMX2 demonstrated slightly reduced expression levels of HMX2 and HMX3, respectively, indicating mutual activation.

    Journal: PLoS ONE

    Article Title: Aberrant expression of NKL homeobox genes HMX2 and HMX3 interferes with cell differentiation in acute myeloid leukemia

    doi: 10.1371/journal.pone.0240120

    Figure Lengend Snippet: Identification of upstream factors regulating HMX2 and HMX3. (A) Treatment of EOL-1 cells with IL7 resulted in increased transcript levels of HMX2 and HMX3 as analyzed by RQ-PCR. Asterisks indicate calculated p-values obtained by t-Test analysis of medium controls and interleukin stimulation. (B) Treatment of EOL-1 and MV4-11 cells with WNT3A and WNT5B resulted in increased expression levels of HMX2 (left) and HMX3 (right). (C) Treatment of EOL-1 cells with NFkB-activator TNFa resulted in reduced expression levels of HMX2 and HMX3 (left). Treatment of EOL-1 cells with NFkB-inhibitor resulted in increased expression of HMX2 and HMX3 (middle). STAT5A served as marker for NFkB-activity which showed low expression levels and increased activity after stimulation with TNFa (right). (D) A genomic map of the locus for HMX2/3 was obtained from the UCSC genome browser, showing potential transcription factor binding sites including those for IRF1, IRF2 and NKX2-5. (E) RQ-PCR analysis of EOL-1 cells treated for siRNA-mediated knockdown of IRF8 resulted in elevated expression levels of HMX2 (left) and HMX3 (right), indicating an activating impact of IRF8. (F) Quantification of IRF8 by RQ-PCR in primary granulocytes and monocytes (left) and selected AML cell lines (right) demonstrated significant levels in EOL-1 and MV4-11. (G) RQ-PCR analysis of EOL-1 (left) and MV4-11 (right) treated for siRNA-mediated knockdown of HMX3 and HMX2 demonstrated slightly reduced expression levels of HMX2 and HMX3, respectively, indicating mutual activation.

    Article Snippet: The results confirmed mutual activation which was more prominent for HMX3 in MV4-11.

    Techniques: Polymerase Chain Reaction, Expressing, Marker, Activity Assay, Binding Assay, Activation Assay

    Regulatory mutations at HMX2/3 in EOL-1. The illustration of RNA-seq data from EOL-1 shows two different mutations at the HMX2/3 locus. (A) An A-to-G mutation in the 5´-region of HMX3 generates a consensus ETS-site. (B) A T-to-C mutation in the 5´-region of HMX2 transforms an NFkB-site into a SP1-site. (C) Quantification of ETS1 (above) and ELK1 (below) in selected AML cell lines by RQ-PCR. (D) Forced expression of ETS1 (above) and ELK1 (below) in EOL-1 cells resulted in respectively reduced and increased expression levels of both HMX2 and HMX3. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (vector) and factor expression. (E) Reporter-gene assay in NIH-3T3 cells using a fragment containing a normal (obtained from MV4-11) or mutated (obtained from EOL-1) ETS-site. Forced expression of ELK1 (left) and ETS1 (right) resulted in elevated reporter-gene activity which was higher using ELK1 for the mutated fragment. (F) Reporter-gene assay in HELA cells using a fragment containing a normal (MV4-11) or mutated (EOL-1) NFkB-site. Treatment with NFkB-activator TNFa (left) resulted in reduced reporter-gene activity which was more pronounced using the normal fragment. Forced expression of SP1 (right) resulted in elevated reporter-gene activity which was higher using the mutated fragment.

    Journal: PLoS ONE

    Article Title: Aberrant expression of NKL homeobox genes HMX2 and HMX3 interferes with cell differentiation in acute myeloid leukemia

    doi: 10.1371/journal.pone.0240120

    Figure Lengend Snippet: Regulatory mutations at HMX2/3 in EOL-1. The illustration of RNA-seq data from EOL-1 shows two different mutations at the HMX2/3 locus. (A) An A-to-G mutation in the 5´-region of HMX3 generates a consensus ETS-site. (B) A T-to-C mutation in the 5´-region of HMX2 transforms an NFkB-site into a SP1-site. (C) Quantification of ETS1 (above) and ELK1 (below) in selected AML cell lines by RQ-PCR. (D) Forced expression of ETS1 (above) and ELK1 (below) in EOL-1 cells resulted in respectively reduced and increased expression levels of both HMX2 and HMX3. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (vector) and factor expression. (E) Reporter-gene assay in NIH-3T3 cells using a fragment containing a normal (obtained from MV4-11) or mutated (obtained from EOL-1) ETS-site. Forced expression of ELK1 (left) and ETS1 (right) resulted in elevated reporter-gene activity which was higher using ELK1 for the mutated fragment. (F) Reporter-gene assay in HELA cells using a fragment containing a normal (MV4-11) or mutated (EOL-1) NFkB-site. Treatment with NFkB-activator TNFa (left) resulted in reduced reporter-gene activity which was more pronounced using the normal fragment. Forced expression of SP1 (right) resulted in elevated reporter-gene activity which was higher using the mutated fragment.

    Article Snippet: The results confirmed mutual activation which was more prominent for HMX3 in MV4-11.

    Techniques: RNA Sequencing Assay, Mutagenesis, Polymerase Chain Reaction, Expressing, Plasmid Preparation, Reporter Gene Assay, Activity Assay

    Regulation of HTR7 by HMX2 and HMX3. (A) Quantification of HTR7 expression in primary granulocytes (neutrophils, eosinophils, basophils), monocytes, and selected cell lines by RQ-PCR. (B) Genomic profiling data demonstrated a duplication in EOL-1 which encompassed the flanking regulatory region of HTR7. (C) A genomic map of the locus for HTR7 was taken from the UCSC genome browser, showing potential transcription factor binding sites. The indicated duplication detected in EOL-1 containes a potential HMX1-site. (D) SiRNA-mediated knockdown of HMX2 (left) and HMX3 (middle) resulted in reduced expression levels of HTR7, indicating an activating impact. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (siCTR) and siRNA-targeted knockdown. Forced expression of HMX2 in HL-60 resulted in elevated transcript levels of HTR7 (right), supporting an activating impact. (E) Reporter-gene assay in NIH-3T3 cells using a fragment containing the identified HMX1-site. Forced expression of HMX2 resulted in decreased reporter-gene activity, demonstrating a direct regulatory impact. (F) Treatment of EOL-1 cells with HTR7-inhibitor MMS and HTR7-activator LP211 resulted in decreased and increased levels of ERK-phosphorylation, respectively, as shown by Western blot. (G) Treatment of EOL-1 cells with HTR7-inhibitor MMS and HTR7-activator LP211 resulted in decreased and increased levels of DLX2-transcription, respectively, as shown by RQ-PCR.

    Journal: PLoS ONE

    Article Title: Aberrant expression of NKL homeobox genes HMX2 and HMX3 interferes with cell differentiation in acute myeloid leukemia

    doi: 10.1371/journal.pone.0240120

    Figure Lengend Snippet: Regulation of HTR7 by HMX2 and HMX3. (A) Quantification of HTR7 expression in primary granulocytes (neutrophils, eosinophils, basophils), monocytes, and selected cell lines by RQ-PCR. (B) Genomic profiling data demonstrated a duplication in EOL-1 which encompassed the flanking regulatory region of HTR7. (C) A genomic map of the locus for HTR7 was taken from the UCSC genome browser, showing potential transcription factor binding sites. The indicated duplication detected in EOL-1 containes a potential HMX1-site. (D) SiRNA-mediated knockdown of HMX2 (left) and HMX3 (middle) resulted in reduced expression levels of HTR7, indicating an activating impact. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (siCTR) and siRNA-targeted knockdown. Forced expression of HMX2 in HL-60 resulted in elevated transcript levels of HTR7 (right), supporting an activating impact. (E) Reporter-gene assay in NIH-3T3 cells using a fragment containing the identified HMX1-site. Forced expression of HMX2 resulted in decreased reporter-gene activity, demonstrating a direct regulatory impact. (F) Treatment of EOL-1 cells with HTR7-inhibitor MMS and HTR7-activator LP211 resulted in decreased and increased levels of ERK-phosphorylation, respectively, as shown by Western blot. (G) Treatment of EOL-1 cells with HTR7-inhibitor MMS and HTR7-activator LP211 resulted in decreased and increased levels of DLX2-transcription, respectively, as shown by RQ-PCR.

    Article Snippet: The results confirmed mutual activation which was more prominent for HMX3 in MV4-11.

    Techniques: Expressing, Polymerase Chain Reaction, Binding Assay, Reporter Gene Assay, Activity Assay, Western Blot

    ISRE induction by ChX710 is dependent on MAVS and IRF1. ( a ) MAVS was silenced by siRNA transfection in ISRE-luciferase reporter cells. After 48 hours of cultures, cells were stimulated with ChX710. After 24 hours of incubation, luciferase induction was determined. ( b ) Same experiment as ( a ) using STING-specific siRNA. ( c ) Same experiment as ( a ) using IRF1-specific siRNA. ( d ) Same experiment as ( a ) using IRF3-specific siRNA. ( e,f ) IRF3 phosphorylation was determined by western-blot in HEK-293T ( e ) or A549 cells ( f ) treated for 24 hours with DMSO alone or ChX710 at 25 μM or 50 μM. ( g ) Schematic model of the signaling pathways induced by ChX710. IRF3 phosphorylation is induced, but this is insufficient to activate IFN-β expression. Data represent means ± SD of three independent experiments, except for ( e ) and ( f ) that corresponds to representative results. ** P

    Journal: Scientific Reports

    Article Title: Identification of a small molecule that primes the type I interferon response to cytosolic DNA

    doi: 10.1038/s41598-017-02776-z

    Figure Lengend Snippet: ISRE induction by ChX710 is dependent on MAVS and IRF1. ( a ) MAVS was silenced by siRNA transfection in ISRE-luciferase reporter cells. After 48 hours of cultures, cells were stimulated with ChX710. After 24 hours of incubation, luciferase induction was determined. ( b ) Same experiment as ( a ) using STING-specific siRNA. ( c ) Same experiment as ( a ) using IRF1-specific siRNA. ( d ) Same experiment as ( a ) using IRF3-specific siRNA. ( e,f ) IRF3 phosphorylation was determined by western-blot in HEK-293T ( e ) or A549 cells ( f ) treated for 24 hours with DMSO alone or ChX710 at 25 μM or 50 μM. ( g ) Schematic model of the signaling pathways induced by ChX710. IRF3 phosphorylation is induced, but this is insufficient to activate IFN-β expression. Data represent means ± SD of three independent experiments, except for ( e ) and ( f ) that corresponds to representative results. ** P

    Article Snippet: Proteins were detected using standard immunoblotting techniques using the following primary antibodies: anti-IRF1 rabbit monoclonal antibody was from Cell Signaling (Clone D5E4; Ref. 8478S), anti-IRF3 rabbit monoclonal antibody was from Cell Signaling (Clone D6I4C; Ref. 11904), anti-P-IRF3 (Ser386) rabbit polyclonal antibody was from Millipore (ABE501), anti-STAT1 mouse monoclonal antibody was from Cell Signaling (Clone 9H2; Ref. 9176), anti-STAT2 rabbit polyclonal antibody was from Cell Signaling (Ref. 4594), anti-MAVS rabbit polyclonal antibody was from Alexis (Ref AT107; 210–929-C100), anti-STING rabbit monoclonal antibody was from Cell Signaling (Clone D2P2F; Ref 13647), anti-ULK1 rabbit monoclonal antibody was from Cell Signaling (Clone D8H5; Ref 8054), and anti-β-Actin mouse monoclonal antibody was from Sigma-Aldrich (Clone AC-15; Ref. A5441).

    Techniques: Transfection, Luciferase, Incubation, Western Blot, Expressing

    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

    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

    CD200 antibody blockade limits tumor progression in GEM model of PDAC. (A) Representative immunofluorescence from pancreatic tissue of KPC-Brca2 mice stained for DAPI (blue), a-SMA (green), and CD200 (red). (B) KPC-Brca2 mice were treated at 6 weeks of age with 200 µg (intraperitoneal injection three times/week) of isotype control or CD200 antibodies (n=5 mice/group). (C) Histology was pathologically scored for PanIN lesions and quantified. Mean±SD; *p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: CD200 promotes immunosuppression in the pancreatic tumor microenvironment

    doi: 10.1136/jitc-2019-000189

    Figure Lengend Snippet: CD200 antibody blockade limits tumor progression in GEM model of PDAC. (A) Representative immunofluorescence from pancreatic tissue of KPC-Brca2 mice stained for DAPI (blue), a-SMA (green), and CD200 (red). (B) KPC-Brca2 mice were treated at 6 weeks of age with 200 µg (intraperitoneal injection three times/week) of isotype control or CD200 antibodies (n=5 mice/group). (C) Histology was pathologically scored for PanIN lesions and quantified. Mean±SD; *p

    Article Snippet: To test whether CD200 affects MDSC activity, we generated MDSC in vitro from healthy donor PBMC stimulated with IL-6 and GM-CSF.

    Techniques: Immunofluorescence, Mouse Assay, Staining, Injection

    CD200 enhances the cytokine-driven differentiation and suppressive activity of MDSC in vitro. Normal donor PBMC were cultured for 7 days with 10 ng/mL of IL-6 and 10 ng/mL of GM-CSF and stained by flow cytometry for the percentage of MDSC (CD11b+CD33+HLA-DR lo ). (A) Representative flow cytometry dot plots from unstimulated or IL-6/GM-CSF stimulated PBMC after 7 days of differentiation. (B) During differentiation, cells were cultured in the presence of recombinant human CD200 protein (rhCD200). (C) Healthy donor PBMC were stimulated with 10 ng/mL of IL-6 and GM-CSF for 30 min with increasing concentrations of rhCD200 protein. Cell lysates were analyzed for STAT3 phosphorylation (p-STAT3) with β-actin as a loading control. (D) Healthy donor PBMC were stimulated with 10 ng/mL of IL-6 and GM-CSF with increasing concentrations of rhCD200 protein for 2 hours. RNA was isolated and expression of IRF-8 was analyzed by real-time PCR. (E) PBMC from healthy donor blood was stimulated with 10 ng/mL of IL-6 and GM-CSF to differentiate cells into MDSC for 5 days. Cells were then cultured with vehicle control or 200 ng/mL rhCD200 for 48 hours. MDSC were then cocultured with autologous CFSE-labeled T cells stimulated. T cells were stimulated with CD3/CD28 beads and proliferation was measured after 4 days by CFSE dilution. (F) Individual donor T-cell proliferation and (G) quantification across all donors. Mean±SD; *p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: CD200 promotes immunosuppression in the pancreatic tumor microenvironment

    doi: 10.1136/jitc-2019-000189

    Figure Lengend Snippet: CD200 enhances the cytokine-driven differentiation and suppressive activity of MDSC in vitro. Normal donor PBMC were cultured for 7 days with 10 ng/mL of IL-6 and 10 ng/mL of GM-CSF and stained by flow cytometry for the percentage of MDSC (CD11b+CD33+HLA-DR lo ). (A) Representative flow cytometry dot plots from unstimulated or IL-6/GM-CSF stimulated PBMC after 7 days of differentiation. (B) During differentiation, cells were cultured in the presence of recombinant human CD200 protein (rhCD200). (C) Healthy donor PBMC were stimulated with 10 ng/mL of IL-6 and GM-CSF for 30 min with increasing concentrations of rhCD200 protein. Cell lysates were analyzed for STAT3 phosphorylation (p-STAT3) with β-actin as a loading control. (D) Healthy donor PBMC were stimulated with 10 ng/mL of IL-6 and GM-CSF with increasing concentrations of rhCD200 protein for 2 hours. RNA was isolated and expression of IRF-8 was analyzed by real-time PCR. (E) PBMC from healthy donor blood was stimulated with 10 ng/mL of IL-6 and GM-CSF to differentiate cells into MDSC for 5 days. Cells were then cultured with vehicle control or 200 ng/mL rhCD200 for 48 hours. MDSC were then cocultured with autologous CFSE-labeled T cells stimulated. T cells were stimulated with CD3/CD28 beads and proliferation was measured after 4 days by CFSE dilution. (F) Individual donor T-cell proliferation and (G) quantification across all donors. Mean±SD; *p

    Article Snippet: To test whether CD200 affects MDSC activity, we generated MDSC in vitro from healthy donor PBMC stimulated with IL-6 and GM-CSF.

    Techniques: Activity Assay, In Vitro, Cell Culture, Staining, Flow Cytometry, Recombinant, Isolation, Expressing, Real-time Polymerase Chain Reaction, Labeling

    CD200 receptor (CD200R) is elevated on MDSC from patients with PDAC. PBMC were isolated from healthy donors (n=9), patients with chronic pancreatitis (CP; n=10), or pancreatic ductal adenocarcinoma (PDAC; n=17) and (A) stained by flow cytometry for granulocytic (CD11b+CD33+HL-DR −/low CD15+) and monocytic (CD11b+CD33+HL-DR -/low CD14+) MDSC. (B) Representative CD200R (blue) or isotype control (red) staining of CD15+ MDSC. (C) Percent total MDSC, (D) CD200R positive cells, and (E) mean fluorescent intensity (MFI) were quantified across all patient groups. (F) Percent and (G) MFI of either CD14+ or CD15+ MDSC that express CD200R. Mean±SD; *p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: CD200 promotes immunosuppression in the pancreatic tumor microenvironment

    doi: 10.1136/jitc-2019-000189

    Figure Lengend Snippet: CD200 receptor (CD200R) is elevated on MDSC from patients with PDAC. PBMC were isolated from healthy donors (n=9), patients with chronic pancreatitis (CP; n=10), or pancreatic ductal adenocarcinoma (PDAC; n=17) and (A) stained by flow cytometry for granulocytic (CD11b+CD33+HL-DR −/low CD15+) and monocytic (CD11b+CD33+HL-DR -/low CD14+) MDSC. (B) Representative CD200R (blue) or isotype control (red) staining of CD15+ MDSC. (C) Percent total MDSC, (D) CD200R positive cells, and (E) mean fluorescent intensity (MFI) were quantified across all patient groups. (F) Percent and (G) MFI of either CD14+ or CD15+ MDSC that express CD200R. Mean±SD; *p

    Article Snippet: To test whether CD200 affects MDSC activity, we generated MDSC in vitro from healthy donor PBMC stimulated with IL-6 and GM-CSF.

    Techniques: Isolation, Staining, Flow Cytometry

    CD200 antibody blockade elicits antitumor response. KPC-derived MT5 tumor were subcutaneously injected into C57BL/6 mice. (A) Mice were treated with 200 µg/mouse of CD200 or isotype control antibodies 3× a week (n=5 mice/group). Intratumoral flow cytometry staining (percent of CD45+ cells and number per mg of tumor tissue) for (B) and (C) MDSC (CD11b+GR1+), (D) and (E) CD4+ T cells, (F) and (G) CD8+ T cells. (H) C57BL/6 mice were inoculated subcutaneously with MT5 tumor cells and were treated once palpable. MT5 tumor-bearing mice were treated with 200 µg/mouse of anti-CD200, PD-1, or isotype control antibodies 3× a week (n=5 mice/group). Mean±SD; * and †=p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: CD200 promotes immunosuppression in the pancreatic tumor microenvironment

    doi: 10.1136/jitc-2019-000189

    Figure Lengend Snippet: CD200 antibody blockade elicits antitumor response. KPC-derived MT5 tumor were subcutaneously injected into C57BL/6 mice. (A) Mice were treated with 200 µg/mouse of CD200 or isotype control antibodies 3× a week (n=5 mice/group). Intratumoral flow cytometry staining (percent of CD45+ cells and number per mg of tumor tissue) for (B) and (C) MDSC (CD11b+GR1+), (D) and (E) CD4+ T cells, (F) and (G) CD8+ T cells. (H) C57BL/6 mice were inoculated subcutaneously with MT5 tumor cells and were treated once palpable. MT5 tumor-bearing mice were treated with 200 µg/mouse of anti-CD200, PD-1, or isotype control antibodies 3× a week (n=5 mice/group). Mean±SD; * and †=p

    Article Snippet: To test whether CD200 affects MDSC activity, we generated MDSC in vitro from healthy donor PBMC stimulated with IL-6 and GM-CSF.

    Techniques: Derivative Assay, Injection, Mouse Assay, Flow Cytometry, Staining

    Pancreatic tumor and stromal cells express elevated levels of CD200. Cell lysates from pancreatic cancer cell lines (BxPC3, MiaPaca2, PANC-1) were analyzed by (A) immunoblot for CD200 with β-actin as a loading control. (B) CD200 surface staining on the pancreatic cancer cell lines were analyzed by flow cytometry (Red, Isotype control; Blue, CD200). (C) Mean fluorescent intensity values from flow cytometry stained cell lines for CD200. (D) Archived surgical patient PDAC specimens were stained by IF for DAPI (blue), α-SMA (green), and CD200 (red). Tumor (Tu) and stromal (S) positive compartments of the tissue are marked in white. α-SMA-alpha-smooth muscle actin.

    Journal: Journal for Immunotherapy of Cancer

    Article Title: CD200 promotes immunosuppression in the pancreatic tumor microenvironment

    doi: 10.1136/jitc-2019-000189

    Figure Lengend Snippet: Pancreatic tumor and stromal cells express elevated levels of CD200. Cell lysates from pancreatic cancer cell lines (BxPC3, MiaPaca2, PANC-1) were analyzed by (A) immunoblot for CD200 with β-actin as a loading control. (B) CD200 surface staining on the pancreatic cancer cell lines were analyzed by flow cytometry (Red, Isotype control; Blue, CD200). (C) Mean fluorescent intensity values from flow cytometry stained cell lines for CD200. (D) Archived surgical patient PDAC specimens were stained by IF for DAPI (blue), α-SMA (green), and CD200 (red). Tumor (Tu) and stromal (S) positive compartments of the tissue are marked in white. α-SMA-alpha-smooth muscle actin.

    Article Snippet: To test whether CD200 affects MDSC activity, we generated MDSC in vitro from healthy donor PBMC stimulated with IL-6 and GM-CSF.

    Techniques: Staining, Flow Cytometry