irf8-mediated inhibition Search Results


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  • 95
    ATCC sendai virus
    Sendai Virus, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore ezh2
    <t>EZH2</t> directly silences IRF8 expression
    Ezh2, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 911 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    86
    Durect Corporation 1002 osmotic minipump
    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.
    1002 Osmotic Minipump, supplied by Durect Corporation, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti caspase 9
    IRF8 depletion suppresses caspase activation and caspase activation is required for EBV lytic replication. A. IRF8 depletion suppresses caspase activation. Western blot analysis of protein extracts from Fig 1D using antibodies against caspase-3, cleaved caspase-3, caspase-8, cleaved caspase-8, caspase-7, cleaved caspase-7, <t>caspase-9,</t> cleaved caspass-9, caspase-2 and Bcl-2 as indicated. B. Caspase inhibition suppresses EBV lytic gene expression. Akata (EBV + ) cells were untreated or pre-treated with pan-caspase inhibitor (Z-VAD-FMK) for 1 hr and then anti-IgG was added for 48 hrs. RNA was extracted and EBV lytic gene expression was analyzed by RT-qPCR. Data are presented as means ± standard deviations of triplicate assays. ** p
    Anti Caspase 9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Cell Signaling Technology Inc irf1
    Regulation of PD-L1 and components of the IFN-γ-signalling cascade in ccRCC cell lines (CaKi-1, A-498), pRCC cell line (CaKi-2) and Cal-54 RCC cell line. a Levels of mRNA (∆Ct) for PD-L1, PD-L2, CXCL10, JAK2, STAT1, <t>IRF1,</t> JAK1, IFN-γR1, IFN-γR2 in control cells (−con) and cells treated with IFN-γ (10 ng/ml) for 24 h (+IFN-γ) are shown. Transcripts that were not inducible by IFN-γ in CaKi-2 cells, in contrast to the other cell lines, are gray-shaded. Box plots indicate means with error bars corresponding to minimum and maximum values ( n = 3). b Western-blot analysis of control cells (con) or cells treated with IFN-γ (10 ng/ml) for 24 h with antibodies for PD-L1, p-JAK2, JAK2, p-JAK1, JAK1, IRF1, and cytoplasmic β-actin. The molecular weights are: PD-L1, ~ 50kd; PD-L2, ~ 50 kd; phosphate (P)-JAK2, 125 kd; JAK2, ~ 125 kd; phosphate (P)-JAK1, 130 kd; JAK1, ~ 130 kd; IRF1, ~ 48 kd; STAT1, ~ 90 kd; β-actin, ~ 43kd. c Schematic diagram of analyzed components of the IFN-γ-signaling cascade. nd below detection level, IFNG IFN-γ, Y tyrosine residue
    Irf1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc caspase 8
    <t>Caspase-8</t> is required for the proteasome-mediated degradation of IRF-3. A , HT1080 cells were transfected with poly(I:C) in the absence or the presence of an inhibitor of caspase-8 (z-IETD, 10 μ m ) for the indicated times, and cell lysates were
    Caspase 8, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 5126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc anti flip antibody
    <t>Caspase-8</t> is required for the proteasome-mediated degradation of IRF-3. A , HT1080 cells were transfected with poly(I:C) in the absence or the presence of an inhibitor of caspase-8 (z-IETD, 10 μ m ) for the indicated times, and cell lysates were
    Anti Flip Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc irf1 d5e4 xp rabbit mab
    Type I IFN induced by STING activation are required for GBP expression that controls bacterial replication BMDMs from C57BL/6, 129/SvEv and STING, cGAS, <t>IRF-1</t> and IFNAR KO mice were infected with B. abortus (MOI 100:1) and total RNA was extracted 17 hrs after infection. Analysis of GBP2 and GBP3 expression by qPCR of macrophages from (A) 129Sv/Ev, IRF-1 and IFNAR KO or (B) C57BL/6, STING and cGAS KO. The results are shown as mean ± SD of fold induction and normalized to β-actin gene. Data are representative of at least three independent experiments. Significant differences comparing WT versus STING or WT versus IFNAR are denoted by an asterisk, WT versus IRF-1 are denoted by #, and STING versus cGAS or IFNAR versus IRF-1 are denoted by (two-way ANOVA, p
    Irf1 D5e4 Xp Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irf1 d5e4 xp rabbit mab/product/Cell Signaling Technology Inc
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    92
    TaKaRa caspase 8
    <t>Caspase</t> 8 and caspase 9 are involved in IRF-3-dependent activation of CPP-32/caspase-3. (A) rtTA-Jurkat IRF-3(5D) cells were cultured in the presence of 4 μg of APO-1-3 per ml or 50 μM Etoposide for 48 h in the continuous presence of caspase blockers zVAD, zIETH, and zLEHD (200 μM), as indicated. Viability was evaluated by using trypan blue exclusion. (B) wtIRF-3-expressing Jurkat cells were treated for 48 h with DOX (5 μg/ml) to stimulate IRF-3 production and were then infected with Sendai virus (400 HAU/ml/10 6 cells) for 24, 48, or 72 h in the continuous presence of caspase blockers zVAD, zIETH, and zLEHD (200 μM). Viability was evaluated by using trypan blue exclusion. (C) IRF-3(5D)-expressing Jurkat cells were treated with DOX (5 μg/ml) for 24, 48, or 72 h to stimulate IRF-3(5D) production. DOX treatment was accomplished in the continuous presence of caspase blockers zVAD, zIETH, and zLEHD (200 μM). Viability was evaluated by using trypan blue exclusion. Symbols in B and C: ■, Sendai virus; ▴, Sendai virus plus zLEHD; ⧫, Sendai virus plus zIETD; ●, Sendai virus plus zVAD. (D) IRF-3(5D)-expressing Jurkat cells were treated with DOX (5 μg/ml) for 24, 48, and 72 h; whole-cell extracts were prepared at different times after treatment with DOX and were incubated with fluorogenic substrates for caspase 8 (■) and caspase 9 (▴) as described in Materials and Methods. Caspase activity is represented by the fluorescence ratio between DOX-induced versus noninduced cells.
    Caspase 8, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
    Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 48620 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bapta am
    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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    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

    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

    IRF8 depletion suppresses caspase activation and caspase activation is required for EBV lytic replication. A. IRF8 depletion suppresses caspase activation. Western blot analysis of protein extracts from Fig 1D using antibodies against caspase-3, cleaved caspase-3, caspase-8, cleaved caspase-8, caspase-7, cleaved caspase-7, caspase-9, cleaved caspass-9, caspase-2 and Bcl-2 as indicated. B. Caspase inhibition suppresses EBV lytic gene expression. Akata (EBV + ) cells were untreated or pre-treated with pan-caspase inhibitor (Z-VAD-FMK) for 1 hr and then anti-IgG was added for 48 hrs. RNA was extracted and EBV lytic gene expression was analyzed by RT-qPCR. Data are presented as means ± standard deviations of triplicate assays. ** p

    Journal: PLoS Pathogens

    Article Title: Interferon regulatory factor 8 regulates caspase-1 expression to facilitate Epstein-Barr virus reactivation in response to B cell receptor stimulation and chemical induction

    doi: 10.1371/journal.ppat.1006868

    Figure Lengend Snippet: IRF8 depletion suppresses caspase activation and caspase activation is required for EBV lytic replication. A. IRF8 depletion suppresses caspase activation. Western blot analysis of protein extracts from Fig 1D using antibodies against caspase-3, cleaved caspase-3, caspase-8, cleaved caspase-8, caspase-7, cleaved caspase-7, caspase-9, cleaved caspass-9, caspase-2 and Bcl-2 as indicated. B. Caspase inhibition suppresses EBV lytic gene expression. Akata (EBV + ) cells were untreated or pre-treated with pan-caspase inhibitor (Z-VAD-FMK) for 1 hr and then anti-IgG was added for 48 hrs. RNA was extracted and EBV lytic gene expression was analyzed by RT-qPCR. Data are presented as means ± standard deviations of triplicate assays. ** p

    Article Snippet: Membranes were then incubated in the following primary antibodies: mouse anti-ZTA (Argene, Cat # 11–007, 1:5,000), mouse anti-RTA (Argene, 1:1,000), mouse anti-BGLF4 antibody (1:1,000) [ ], anti-β-actin (Sigma, Cat # A5441, 1:5,000), anti-IRF8 (CST, Cat #5628, 1:1,000), anti-PARP (CST, Cat #9532, 1:1,000), anti-Cleaved PARP (CST, Cat #5625, 1:1,000), anti-Cleaved Caspase Substrates (CST, Cat #8698, 1:1,000), anti-Caspase-1 (CST, Cat #3866, 1:1,000), anti-Caspase-2 (CST, Cat #2224, 1:1,000), anti-Caspase-3 (Santa Cruz, Cat #sc-7148, 1:1,000), anti-Cleaved Caspase-3 (CST, Cat #9664, 1:1,000), anti-Caspase-7 (CST, Cat #12827, 1:1,000), anti-Cleaved Caspase-7 (CST, Cat #8438, 1:1,000), anti-Caspase-8 (CST, Cat #9746, 1:1,000), anti-Cleaved Caspase-8 (CST, Cat #9496, 1:1,000), anti-Caspase-9 (CST, Cat #9508, 1:1,000), anti-Bcl-2 (Bethyl, Cat #A303-675A, 1:1,000), anti-KAP1 (CST, Cat #4123, 1:1,000), anti-PAX5 (CST, Cat #8970, 1:1,000), anti-DNMT3A (Bethyl, Cat #A304-278A, 1:1,000), anti-STAT3 (CST, Cat #9139, 1:1,000), and anti-HA (CST, Cat #14031S, 1:1,000).

    Techniques: Activation Assay, Western Blot, Inhibition, Expressing, Quantitative RT-PCR

    Regulation of PD-L1 and components of the IFN-γ-signalling cascade in ccRCC cell lines (CaKi-1, A-498), pRCC cell line (CaKi-2) and Cal-54 RCC cell line. a Levels of mRNA (∆Ct) for PD-L1, PD-L2, CXCL10, JAK2, STAT1, IRF1, JAK1, IFN-γR1, IFN-γR2 in control cells (−con) and cells treated with IFN-γ (10 ng/ml) for 24 h (+IFN-γ) are shown. Transcripts that were not inducible by IFN-γ in CaKi-2 cells, in contrast to the other cell lines, are gray-shaded. Box plots indicate means with error bars corresponding to minimum and maximum values ( n = 3). b Western-blot analysis of control cells (con) or cells treated with IFN-γ (10 ng/ml) for 24 h with antibodies for PD-L1, p-JAK2, JAK2, p-JAK1, JAK1, IRF1, and cytoplasmic β-actin. The molecular weights are: PD-L1, ~ 50kd; PD-L2, ~ 50 kd; phosphate (P)-JAK2, 125 kd; JAK2, ~ 125 kd; phosphate (P)-JAK1, 130 kd; JAK1, ~ 130 kd; IRF1, ~ 48 kd; STAT1, ~ 90 kd; β-actin, ~ 43kd. c Schematic diagram of analyzed components of the IFN-γ-signaling cascade. nd below detection level, IFNG IFN-γ, Y tyrosine residue

    Journal: Targeted Oncology

    Article Title: Co-Regulation of Immune Checkpoint PD-L1 with Interferon-Gamma Signaling is Associated with a Survival Benefit in Renal Cell Cancer

    doi: 10.1007/s11523-020-00728-8

    Figure Lengend Snippet: Regulation of PD-L1 and components of the IFN-γ-signalling cascade in ccRCC cell lines (CaKi-1, A-498), pRCC cell line (CaKi-2) and Cal-54 RCC cell line. a Levels of mRNA (∆Ct) for PD-L1, PD-L2, CXCL10, JAK2, STAT1, IRF1, JAK1, IFN-γR1, IFN-γR2 in control cells (−con) and cells treated with IFN-γ (10 ng/ml) for 24 h (+IFN-γ) are shown. Transcripts that were not inducible by IFN-γ in CaKi-2 cells, in contrast to the other cell lines, are gray-shaded. Box plots indicate means with error bars corresponding to minimum and maximum values ( n = 3). b Western-blot analysis of control cells (con) or cells treated with IFN-γ (10 ng/ml) for 24 h with antibodies for PD-L1, p-JAK2, JAK2, p-JAK1, JAK1, IRF1, and cytoplasmic β-actin. The molecular weights are: PD-L1, ~ 50kd; PD-L2, ~ 50 kd; phosphate (P)-JAK2, 125 kd; JAK2, ~ 125 kd; phosphate (P)-JAK1, 130 kd; JAK1, ~ 130 kd; IRF1, ~ 48 kd; STAT1, ~ 90 kd; β-actin, ~ 43kd. c Schematic diagram of analyzed components of the IFN-γ-signaling cascade. nd below detection level, IFNG IFN-γ, Y tyrosine residue

    Article Snippet: The membranes were blocked at room temperature for 1.5 h in TRIS-buffered saline with 0.1% Tween containing 5% dry milk, and then the primary antibodies were added and incubated at 4 °C for 24–48 h. The antibodies were as follows: PD-L1 #13684, PD-L2 #82723, STAT1 #9172, JAK1 #3344, phospho-JAK1 #74129, IRF1 #8478, JAK2 #3230, phospho-JAK2 #3771; host rabbit (Cell Signalling Technology Europe, Frankfurt a.M., Germany); β-actin (#MAK6019); host mouse (Linaris, Dossenheim, Germany).

    Techniques: Western Blot

    ConA-induced hepatic injury in IFNαβR-KO mice. A, mice were euthanized 6 ( WT-B6 ) or 8 h ( WT-BALB/c or IFN αβ R-KO ) after administering 20 mg/kg of ConA. Representative sections of the vehicle- or ConA-treated livers stained with hematoxylin/eosin (H/E) show protection in IFNαβR-KO mice compared with WT controls. Bar graph shows quantification of necrotic area as defined in the H/E-stained liver sections. Scale bars , 100 μm ( left and middle panels ) and 50 μm ( right panels ). B, mice were euthanized 1, 3, or 6 h after administering 20 mg/kg of ConA. ConA-treated IFNαβR-KO mice show significantly lower serum ALT levels as compared with the WT mice. Data are expressed as mean ± S.D. n = 4–6 mice/group. C, Western blotting of the nuclear lysates shows IRF1 in WT ( B6 and BALB/c ) and IFNαβR-KO mice. Bar graph shows the relative densitometry values normalized to TBP as an internal control ( n = 4–6 mice/group). D, mRNA expression of the indicated mediators in ConA-challenged mice at 1, 3, or 6 h as measured via qRT-PCR. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Mechanisms of concanavalin A-induced cytokine synthesis by hepatic stellate cells: Distinct roles of interferon regulatory factor-1 in liver injury

    doi: 10.1074/jbc.RA118.005583

    Figure Lengend Snippet: ConA-induced hepatic injury in IFNαβR-KO mice. A, mice were euthanized 6 ( WT-B6 ) or 8 h ( WT-BALB/c or IFN αβ R-KO ) after administering 20 mg/kg of ConA. Representative sections of the vehicle- or ConA-treated livers stained with hematoxylin/eosin (H/E) show protection in IFNαβR-KO mice compared with WT controls. Bar graph shows quantification of necrotic area as defined in the H/E-stained liver sections. Scale bars , 100 μm ( left and middle panels ) and 50 μm ( right panels ). B, mice were euthanized 1, 3, or 6 h after administering 20 mg/kg of ConA. ConA-treated IFNαβR-KO mice show significantly lower serum ALT levels as compared with the WT mice. Data are expressed as mean ± S.D. n = 4–6 mice/group. C, Western blotting of the nuclear lysates shows IRF1 in WT ( B6 and BALB/c ) and IFNαβR-KO mice. Bar graph shows the relative densitometry values normalized to TBP as an internal control ( n = 4–6 mice/group). D, mRNA expression of the indicated mediators in ConA-challenged mice at 1, 3, or 6 h as measured via qRT-PCR. *, p

    Article Snippet: The reagents were obtained from the indicated sources: concanavalin A, d -mannose, Me-αMan, protease, Nycodenz, collagenase (C5138), and BMS-345541 (Sigma); FITC-conjugated ConA (FITC-ConA) (Vector Laboratories, Burlingame, CA); collagenase Type 4 (Worthington Biochemical Corp.); LY294002, AG490, PP2, SB202190, GSK690693, SR11302, and fludarabine (Tocris Bioscience, Minneapolis, MN); PD98059 and SP600125 (Calbiochem); SN50 (BioVision, Inc., Milpitas, CA); anti-P-JAK2 (Tyk2), anti-P-STAT1, anti-IRF1, anti-P-JNK1/2, anti-cleaved caspase 3, anti-Histone H3, and anti-TBP Abs (Cell Signaling, Danvers, MA); horseradish peroxidase-conjugated β-actin Ab (Santa Cruz Biotechnology, Inc.); rabbit anti-GFAP (Dako Cytomation, Carpinteria, CA); MitoTracker Red and anti-desmin monoclonal Ab (DE-U-10) (Life Technologies Corp.).

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

    Hepatocytes from IRF1-KO and IFNαβR-KO are resistant to oxidative stress and apoptosis by ConA-stimulated WT-HSCs. A–D, hepatocytes ( WT or IRF1-KO or IFN αβ R-KO ) were incubated in medium without or with 50 μg/ml of ConA or medium conditioned by HSCs (WT or IRF1-KO) for 8 h in the absence or presence of ConA. After 12 h of incubation, DCFDA staining was performed to determine oxidative stress in hepatocytes. An overlay of phase-contrast and DCF image in A shows ROS generation and bleb formation in WT hepatocytes treated with ConA/HSC medium from WT mice. Scale bars , 20 μm. Bar graph ( E ) shows mean fluorescence intensity ( MFI for phase-contrast images). F, mRNA expression of SOD1 in WT hepatocytes incubated in medium without or with 50 μg/ml of ConA or medium conditioned by WT-HSCs. The data are representative of three sets of experiments. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Mechanisms of concanavalin A-induced cytokine synthesis by hepatic stellate cells: Distinct roles of interferon regulatory factor-1 in liver injury

    doi: 10.1074/jbc.RA118.005583

    Figure Lengend Snippet: Hepatocytes from IRF1-KO and IFNαβR-KO are resistant to oxidative stress and apoptosis by ConA-stimulated WT-HSCs. A–D, hepatocytes ( WT or IRF1-KO or IFN αβ R-KO ) were incubated in medium without or with 50 μg/ml of ConA or medium conditioned by HSCs (WT or IRF1-KO) for 8 h in the absence or presence of ConA. After 12 h of incubation, DCFDA staining was performed to determine oxidative stress in hepatocytes. An overlay of phase-contrast and DCF image in A shows ROS generation and bleb formation in WT hepatocytes treated with ConA/HSC medium from WT mice. Scale bars , 20 μm. Bar graph ( E ) shows mean fluorescence intensity ( MFI for phase-contrast images). F, mRNA expression of SOD1 in WT hepatocytes incubated in medium without or with 50 μg/ml of ConA or medium conditioned by WT-HSCs. The data are representative of three sets of experiments. *, p

    Article Snippet: The reagents were obtained from the indicated sources: concanavalin A, d -mannose, Me-αMan, protease, Nycodenz, collagenase (C5138), and BMS-345541 (Sigma); FITC-conjugated ConA (FITC-ConA) (Vector Laboratories, Burlingame, CA); collagenase Type 4 (Worthington Biochemical Corp.); LY294002, AG490, PP2, SB202190, GSK690693, SR11302, and fludarabine (Tocris Bioscience, Minneapolis, MN); PD98059 and SP600125 (Calbiochem); SN50 (BioVision, Inc., Milpitas, CA); anti-P-JAK2 (Tyk2), anti-P-STAT1, anti-IRF1, anti-P-JNK1/2, anti-cleaved caspase 3, anti-Histone H3, and anti-TBP Abs (Cell Signaling, Danvers, MA); horseradish peroxidase-conjugated β-actin Ab (Santa Cruz Biotechnology, Inc.); rabbit anti-GFAP (Dako Cytomation, Carpinteria, CA); MitoTracker Red and anti-desmin monoclonal Ab (DE-U-10) (Life Technologies Corp.).

    Techniques: Incubation, Staining, Mouse Assay, Fluorescence, Expressing

    ConA does not induce expression of cytokines in IRF1-KO HSCs despite stimulating JAK/STAT signaling. A, HSCs from WT mice were stimulated with 50 μg/ml of ConA for 6 h and localization of IRF-1 ( red ) was determined by immunostaining with anti-IRF1 Ab. Nuclear IRF1 is strongly increased in ConA-stimulated HSCs. Nuclei are stained with DAPI ( blue ). Scale bars , 20 μm. B, mRNA expression of the indicated mediators in HSCs from WT or IRF1-KO mice stimulated with 50 μg/ml of ConA for 1, 3, or 6 h as measured via qRT-PCR. C, Western blotting shows P-JAK2 (cytosolic) and P-STAT1 (nuclear) expression in protein lysates prepared from ConA-stimulated IRF1-KO HSCs for the indicated time points. Bar graph shows the relative densitometry values normalized to respective internal controls. All data are representative of three independent experiments. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Mechanisms of concanavalin A-induced cytokine synthesis by hepatic stellate cells: Distinct roles of interferon regulatory factor-1 in liver injury

    doi: 10.1074/jbc.RA118.005583

    Figure Lengend Snippet: ConA does not induce expression of cytokines in IRF1-KO HSCs despite stimulating JAK/STAT signaling. A, HSCs from WT mice were stimulated with 50 μg/ml of ConA for 6 h and localization of IRF-1 ( red ) was determined by immunostaining with anti-IRF1 Ab. Nuclear IRF1 is strongly increased in ConA-stimulated HSCs. Nuclei are stained with DAPI ( blue ). Scale bars , 20 μm. B, mRNA expression of the indicated mediators in HSCs from WT or IRF1-KO mice stimulated with 50 μg/ml of ConA for 1, 3, or 6 h as measured via qRT-PCR. C, Western blotting shows P-JAK2 (cytosolic) and P-STAT1 (nuclear) expression in protein lysates prepared from ConA-stimulated IRF1-KO HSCs for the indicated time points. Bar graph shows the relative densitometry values normalized to respective internal controls. All data are representative of three independent experiments. *, p

    Article Snippet: The reagents were obtained from the indicated sources: concanavalin A, d -mannose, Me-αMan, protease, Nycodenz, collagenase (C5138), and BMS-345541 (Sigma); FITC-conjugated ConA (FITC-ConA) (Vector Laboratories, Burlingame, CA); collagenase Type 4 (Worthington Biochemical Corp.); LY294002, AG490, PP2, SB202190, GSK690693, SR11302, and fludarabine (Tocris Bioscience, Minneapolis, MN); PD98059 and SP600125 (Calbiochem); SN50 (BioVision, Inc., Milpitas, CA); anti-P-JAK2 (Tyk2), anti-P-STAT1, anti-IRF1, anti-P-JNK1/2, anti-cleaved caspase 3, anti-Histone H3, and anti-TBP Abs (Cell Signaling, Danvers, MA); horseradish peroxidase-conjugated β-actin Ab (Santa Cruz Biotechnology, Inc.); rabbit anti-GFAP (Dako Cytomation, Carpinteria, CA); MitoTracker Red and anti-desmin monoclonal Ab (DE-U-10) (Life Technologies Corp.).

    Techniques: Expressing, Mouse Assay, Immunostaining, Staining, Quantitative RT-PCR, Western Blot

    ConA-induced hepatic expression of IFNγ and SOD1. Mice were euthanized 6 h (WT-B6 or IRF1-KO) or 8 h (WT-BALB/c or IFNαβR-KO) after administering 20 mg/kg of ConA. Hepatic mRNA expression of ( A ) IFNγ and ( B ) SOD1 is shown ( n = 6–8 mice/group). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Mechanisms of concanavalin A-induced cytokine synthesis by hepatic stellate cells: Distinct roles of interferon regulatory factor-1 in liver injury

    doi: 10.1074/jbc.RA118.005583

    Figure Lengend Snippet: ConA-induced hepatic expression of IFNγ and SOD1. Mice were euthanized 6 h (WT-B6 or IRF1-KO) or 8 h (WT-BALB/c or IFNαβR-KO) after administering 20 mg/kg of ConA. Hepatic mRNA expression of ( A ) IFNγ and ( B ) SOD1 is shown ( n = 6–8 mice/group). *, p

    Article Snippet: The reagents were obtained from the indicated sources: concanavalin A, d -mannose, Me-αMan, protease, Nycodenz, collagenase (C5138), and BMS-345541 (Sigma); FITC-conjugated ConA (FITC-ConA) (Vector Laboratories, Burlingame, CA); collagenase Type 4 (Worthington Biochemical Corp.); LY294002, AG490, PP2, SB202190, GSK690693, SR11302, and fludarabine (Tocris Bioscience, Minneapolis, MN); PD98059 and SP600125 (Calbiochem); SN50 (BioVision, Inc., Milpitas, CA); anti-P-JAK2 (Tyk2), anti-P-STAT1, anti-IRF1, anti-P-JNK1/2, anti-cleaved caspase 3, anti-Histone H3, and anti-TBP Abs (Cell Signaling, Danvers, MA); horseradish peroxidase-conjugated β-actin Ab (Santa Cruz Biotechnology, Inc.); rabbit anti-GFAP (Dako Cytomation, Carpinteria, CA); MitoTracker Red and anti-desmin monoclonal Ab (DE-U-10) (Life Technologies Corp.).

    Techniques: Expressing, Mouse Assay

    ConA-stimulated WT-HSCs do not stimulate JNK or caspase 3 activation in hepatocytes from IRF1-KO and IFNαβR-KO mice. A, for densitometry values.) B, WT hepatocytes were incubated for 12 h without/with ConA or medium conditioned by WT-HSCs in the absence or presence of ConA ± JNK inhibitor SP600125 ( JNKi ; 10 μg/ml) or anti-IFNβ Ab (2 μg/ml). SOD1 mRNA expression and SOD activity were determined. C, cytosolic and nuclear lysates prepared from hepatocytes in B were analyzed by Western blotting. Bar graph shows relative densitometry values normalized to the respective internal controls. All data are representative of three independent experiments. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Mechanisms of concanavalin A-induced cytokine synthesis by hepatic stellate cells: Distinct roles of interferon regulatory factor-1 in liver injury

    doi: 10.1074/jbc.RA118.005583

    Figure Lengend Snippet: ConA-stimulated WT-HSCs do not stimulate JNK or caspase 3 activation in hepatocytes from IRF1-KO and IFNαβR-KO mice. A, for densitometry values.) B, WT hepatocytes were incubated for 12 h without/with ConA or medium conditioned by WT-HSCs in the absence or presence of ConA ± JNK inhibitor SP600125 ( JNKi ; 10 μg/ml) or anti-IFNβ Ab (2 μg/ml). SOD1 mRNA expression and SOD activity were determined. C, cytosolic and nuclear lysates prepared from hepatocytes in B were analyzed by Western blotting. Bar graph shows relative densitometry values normalized to the respective internal controls. All data are representative of three independent experiments. *, p

    Article Snippet: The reagents were obtained from the indicated sources: concanavalin A, d -mannose, Me-αMan, protease, Nycodenz, collagenase (C5138), and BMS-345541 (Sigma); FITC-conjugated ConA (FITC-ConA) (Vector Laboratories, Burlingame, CA); collagenase Type 4 (Worthington Biochemical Corp.); LY294002, AG490, PP2, SB202190, GSK690693, SR11302, and fludarabine (Tocris Bioscience, Minneapolis, MN); PD98059 and SP600125 (Calbiochem); SN50 (BioVision, Inc., Milpitas, CA); anti-P-JAK2 (Tyk2), anti-P-STAT1, anti-IRF1, anti-P-JNK1/2, anti-cleaved caspase 3, anti-Histone H3, and anti-TBP Abs (Cell Signaling, Danvers, MA); horseradish peroxidase-conjugated β-actin Ab (Santa Cruz Biotechnology, Inc.); rabbit anti-GFAP (Dako Cytomation, Carpinteria, CA); MitoTracker Red and anti-desmin monoclonal Ab (DE-U-10) (Life Technologies Corp.).

    Techniques: Activation Assay, Mouse Assay, Incubation, Expressing, Activity Assay, Western Blot

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep24409

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

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

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

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep24409

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

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

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

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep24409

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

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

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

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

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

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

    doi: 10.1152/ajplung.00368.2013

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

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

    Techniques: Activation Assay, Inhibition

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

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

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

    doi: 10.1152/ajplung.00368.2013

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

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

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

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

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

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

    doi: 10.1152/ajplung.00368.2013

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

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

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

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

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

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

    doi: 10.1152/ajplung.00368.2013

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

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

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

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.9365

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

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

    Techniques:

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.9365

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

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

    Techniques: Activation Assay, Western Blot

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.9365

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

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

    Techniques:

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.9365

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

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

    Techniques: Transfection

    IFN-γ-stimulated expression of APOL1 in differentiated U937 monocytes contributes to inhibition of HIV-1 production. (A) APOL1 expression is inducible by IFN-γ in differentiated human monocytes (THP-1 and U937) but not in CD4 + T cells (SupT1 and Jurkat). The cells were stimulated for 24 h with PMA (50 ng/ml) or were left untreated (control). After a washing, PMA-treated cells were cultured in media alone (PMA) or were treated for another 24 h with IFN-γ (20 ng/ml) (PMA/IFN-γ). As an additional control, 293T cells were transfected with APOL1 vector (0.5 μg). Cell lysates (50 μg/lane) were analyzed by immunoblotting for the expression of APOL1 and actin. (B) ( ). (Right side) Seven-day differentiated MDM were either left untreated (control) or stimulated for 18 h with IFN-α or IFN-γ at 100 ng/ml. Cell lysate proteins were resolved by 4 to 12% gradient SDS-PAGE, and the levels of APOL1, IRF-1, A3A, and Samhd1 were analyzed by Western blotting. (C) U937 clones expressing control shRNA (control) or APOL1 shRNA (APOL1) were stimulated for 24 h with PMA (50 ng/ml), followed by treatment with IFN-γ (20 ng/ml) as indicated. After 24 h, the cells were rinsed and exposed to HIV-1 89.6 (50 ng of p24 per 10 6 cells) pseudotyped with VSV-G. After 4 h of incubation, the cells were washed and cultivated for another 48 h, followed by collection of supernatants and cell lysates. Pelleted virus and lysates were analyzed by immunoblotting for the expression of HIV-1 Gag, APOL1, and actin. Virus production was determined by the expression of pelletable p24. Protein signals were obtained from densitometric scanning. Virus released from PMA-treated U937-CoshRNA cells (control) was used as a reference (100%).

    Journal: Journal of Virology

    Article Title: The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection

    doi: 10.1128/JVI.02828-13

    Figure Lengend Snippet: IFN-γ-stimulated expression of APOL1 in differentiated U937 monocytes contributes to inhibition of HIV-1 production. (A) APOL1 expression is inducible by IFN-γ in differentiated human monocytes (THP-1 and U937) but not in CD4 + T cells (SupT1 and Jurkat). The cells were stimulated for 24 h with PMA (50 ng/ml) or were left untreated (control). After a washing, PMA-treated cells were cultured in media alone (PMA) or were treated for another 24 h with IFN-γ (20 ng/ml) (PMA/IFN-γ). As an additional control, 293T cells were transfected with APOL1 vector (0.5 μg). Cell lysates (50 μg/lane) were analyzed by immunoblotting for the expression of APOL1 and actin. (B) ( ). (Right side) Seven-day differentiated MDM were either left untreated (control) or stimulated for 18 h with IFN-α or IFN-γ at 100 ng/ml. Cell lysate proteins were resolved by 4 to 12% gradient SDS-PAGE, and the levels of APOL1, IRF-1, A3A, and Samhd1 were analyzed by Western blotting. (C) U937 clones expressing control shRNA (control) or APOL1 shRNA (APOL1) were stimulated for 24 h with PMA (50 ng/ml), followed by treatment with IFN-γ (20 ng/ml) as indicated. After 24 h, the cells were rinsed and exposed to HIV-1 89.6 (50 ng of p24 per 10 6 cells) pseudotyped with VSV-G. After 4 h of incubation, the cells were washed and cultivated for another 48 h, followed by collection of supernatants and cell lysates. Pelleted virus and lysates were analyzed by immunoblotting for the expression of HIV-1 Gag, APOL1, and actin. Virus production was determined by the expression of pelletable p24. Protein signals were obtained from densitometric scanning. Virus released from PMA-treated U937-CoshRNA cells (control) was used as a reference (100%).

    Article Snippet: The following antibodies were used: anti-Eps15, anti-Rab7, anti-lysosome-associated membrane protein 1 (anti-LAMP1), and anti-IRF-1 (Cell Signaling); anti-Stx7 (R & D Systems); anti-VAMP7 (ProSci); anti-Stx17 (Abgent), anti-APOL1, antiactin, and antitubulin (Sigma-Aldrich); anti-Alix, anti-CD81, anti-CD63, and anti-APOBEC3A (Santa Cruz Biotechnology); anti-Samhd1 (Abcam); and anti-Tsg101 (GeneTex).

    Techniques: Expressing, Inhibition, Cell Culture, Transfection, Plasmid Preparation, SDS Page, Western Blot, Clone Assay, shRNA, Incubation

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

    Journal: Oncology Reports

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

    doi: 10.3892/or.2016.4864

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

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

    Techniques: Expressing, Real-time Polymerase Chain Reaction

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

    Journal: Oncology Reports

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

    doi: 10.3892/or.2016.4864

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

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

    Techniques: Expressing

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

    Journal: Oncology Reports

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

    doi: 10.3892/or.2016.4864

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

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

    Techniques: Staining, Expressing, Negative Control, Infection

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

    Journal: Oncology Reports

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

    doi: 10.3892/or.2016.4864

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

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

    Techniques: Binding Assay, Luciferase, Activity Assay

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

    Journal: Oncology Reports

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

    doi: 10.3892/or.2016.4864

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

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

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

    Transcriptomic analyses of selected TF families in TLR4-primed hMSCs. ( A ) Heat map representing differential expression of NF-κB TF families IRF1, and IRF7. ( B ) UCSC Genome browser images representing normalized RNA-seq read densities for TF expression in TLR4-primed vs. control hMSCs. ( C ) Confirmation of differentially expressed TFs by quantitative reverse transcription-polymerase chain reaction and western blotting in TLR4-primed hMSCs (* P

    Journal: Scientific Reports

    Article Title: Transcriptome sequencing wide functional analysis of human mesenchymal stem cells in response to TLR4 ligand

    doi: 10.1038/srep30311

    Figure Lengend Snippet: Transcriptomic analyses of selected TF families in TLR4-primed hMSCs. ( A ) Heat map representing differential expression of NF-κB TF families IRF1, and IRF7. ( B ) UCSC Genome browser images representing normalized RNA-seq read densities for TF expression in TLR4-primed vs. control hMSCs. ( C ) Confirmation of differentially expressed TFs by quantitative reverse transcription-polymerase chain reaction and western blotting in TLR4-primed hMSCs (* P

    Article Snippet: After sonication, the cells were incubated with RIPA buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 0.1% SDS, 0.1% Sodium deoxycholate, 1% Triton X-100) and subjected to immunoprecipitation with antibodies against IRF1 (Cell Signaling, Danvers, MA; #8478) and normal rabbit IgG (Santa Cruz Biotechnology, Inc., Dallas, TX; sc#2025) used as a control with Dynabeads Protein A beads (Invitrogen, Waltham, MA) for 16 hr at 4 °C.

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

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M111.257022

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

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

    Techniques: Transfection

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M111.257022

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

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

    Techniques: Infection, Activation Assay, Western Blot

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M111.257022

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

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

    Techniques: Activity Assay, Expressing

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M111.257022

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

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

    Techniques:

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M111.257022

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

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

    Techniques: Mutagenesis, Expressing, Transfection

    Type I IFN induced by STING activation are required for GBP expression that controls bacterial replication BMDMs from C57BL/6, 129/SvEv and STING, cGAS, IRF-1 and IFNAR KO mice were infected with B. abortus (MOI 100:1) and total RNA was extracted 17 hrs after infection. Analysis of GBP2 and GBP3 expression by qPCR of macrophages from (A) 129Sv/Ev, IRF-1 and IFNAR KO or (B) C57BL/6, STING and cGAS KO. The results are shown as mean ± SD of fold induction and normalized to β-actin gene. Data are representative of at least three independent experiments. Significant differences comparing WT versus STING or WT versus IFNAR are denoted by an asterisk, WT versus IRF-1 are denoted by #, and STING versus cGAS or IFNAR versus IRF-1 are denoted by (two-way ANOVA, p

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

    Article Title: Brucella abortus triggers a cGAS-independent STING pathway to induce host protection that involves guanylate-binding proteins and inflammasome activation

    doi: 10.4049/jimmunol.1700725

    Figure Lengend Snippet: Type I IFN induced by STING activation are required for GBP expression that controls bacterial replication BMDMs from C57BL/6, 129/SvEv and STING, cGAS, IRF-1 and IFNAR KO mice were infected with B. abortus (MOI 100:1) and total RNA was extracted 17 hrs after infection. Analysis of GBP2 and GBP3 expression by qPCR of macrophages from (A) 129Sv/Ev, IRF-1 and IFNAR KO or (B) C57BL/6, STING and cGAS KO. The results are shown as mean ± SD of fold induction and normalized to β-actin gene. Data are representative of at least three independent experiments. Significant differences comparing WT versus STING or WT versus IFNAR are denoted by an asterisk, WT versus IRF-1 are denoted by #, and STING versus cGAS or IFNAR versus IRF-1 are denoted by (two-way ANOVA, p

    Article Snippet: The following primary antibodies were incubated overnight at 4°C: rabbit mAb IRF-1 #8478 (Cell Signaling); rabbit mAb β-Actin #4970 (Cell Signaling) anti-Caspase-1 (p20, mouse mAb #AG-20B-0042, Adipogen) and IL-1β (mouse mAb #3A6, Cell Signaling).

    Techniques: Activation Assay, Expressing, Mouse Assay, Infection, Real-time Polymerase Chain Reaction

    IRF-1 and type I IFN signaling are required to restrict Brucella replication in macrophages Macrophages derived from (A) C57BL/6, STING, cGAS and AIM2 KO or (B) 129Sv/Ev and IFNAR KO mice were infected with B. abortus (at MOI 100:1) or transfected with bacterial DNA (1μg/well) or poly dA:dT (1μg/well) encapsulated with lipofectamine or lipofectamine alone as control. Cell lysates were harvested 17 hrs after treatment and processing by western blot to determine the levels of IRF-1. (C) BMDMs derived from WT (129 Sv/Ev), IFNAR −/− , or IRF-1 −/− mice were infected with Brucella -GFP (MOI 10:1) for 24 hrs and processed for fluorescent microscopy analysis. GFP-expressing bacteria are shown in green, phalloidin staining of the actin cytoskeleton for cell shape determination is shown in red, and DAPI (DNA) is shown in blue. Images show infected 129Sv/Ev, IFNAR −/− , or IRF-1 −/− macrophages on top, middle and lower panels, respectively, as indicated on the left. Size bar shown on the lower-right panel in B corresponds to 30 μm in all panels. (D) The number of GFP-expressing bacteria was assessed for each cell, and 200 cells were analyzed for each mouse strain. Significant differences in average number of Brucella /cell comparing WT versus IFNAR and IRF-1 KO is denoted by *** (p

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

    Article Title: Brucella abortus triggers a cGAS-independent STING pathway to induce host protection that involves guanylate-binding proteins and inflammasome activation

    doi: 10.4049/jimmunol.1700725

    Figure Lengend Snippet: IRF-1 and type I IFN signaling are required to restrict Brucella replication in macrophages Macrophages derived from (A) C57BL/6, STING, cGAS and AIM2 KO or (B) 129Sv/Ev and IFNAR KO mice were infected with B. abortus (at MOI 100:1) or transfected with bacterial DNA (1μg/well) or poly dA:dT (1μg/well) encapsulated with lipofectamine or lipofectamine alone as control. Cell lysates were harvested 17 hrs after treatment and processing by western blot to determine the levels of IRF-1. (C) BMDMs derived from WT (129 Sv/Ev), IFNAR −/− , or IRF-1 −/− mice were infected with Brucella -GFP (MOI 10:1) for 24 hrs and processed for fluorescent microscopy analysis. GFP-expressing bacteria are shown in green, phalloidin staining of the actin cytoskeleton for cell shape determination is shown in red, and DAPI (DNA) is shown in blue. Images show infected 129Sv/Ev, IFNAR −/− , or IRF-1 −/− macrophages on top, middle and lower panels, respectively, as indicated on the left. Size bar shown on the lower-right panel in B corresponds to 30 μm in all panels. (D) The number of GFP-expressing bacteria was assessed for each cell, and 200 cells were analyzed for each mouse strain. Significant differences in average number of Brucella /cell comparing WT versus IFNAR and IRF-1 KO is denoted by *** (p

    Article Snippet: The following primary antibodies were incubated overnight at 4°C: rabbit mAb IRF-1 #8478 (Cell Signaling); rabbit mAb β-Actin #4970 (Cell Signaling) anti-Caspase-1 (p20, mouse mAb #AG-20B-0042, Adipogen) and IL-1β (mouse mAb #3A6, Cell Signaling).

    Techniques: Derivative Assay, Mouse Assay, Infection, Transfection, Western Blot, Microscopy, Expressing, Staining

    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

    Caspase 8 and caspase 9 are involved in IRF-3-dependent activation of CPP-32/caspase-3. (A) rtTA-Jurkat IRF-3(5D) cells were cultured in the presence of 4 μg of APO-1-3 per ml or 50 μM Etoposide for 48 h in the continuous presence of caspase blockers zVAD, zIETH, and zLEHD (200 μM), as indicated. Viability was evaluated by using trypan blue exclusion. (B) wtIRF-3-expressing Jurkat cells were treated for 48 h with DOX (5 μg/ml) to stimulate IRF-3 production and were then infected with Sendai virus (400 HAU/ml/10 6 cells) for 24, 48, or 72 h in the continuous presence of caspase blockers zVAD, zIETH, and zLEHD (200 μM). Viability was evaluated by using trypan blue exclusion. (C) IRF-3(5D)-expressing Jurkat cells were treated with DOX (5 μg/ml) for 24, 48, or 72 h to stimulate IRF-3(5D) production. DOX treatment was accomplished in the continuous presence of caspase blockers zVAD, zIETH, and zLEHD (200 μM). Viability was evaluated by using trypan blue exclusion. Symbols in B and C: ■, Sendai virus; ▴, Sendai virus plus zLEHD; ⧫, Sendai virus plus zIETD; ●, Sendai virus plus zVAD. (D) IRF-3(5D)-expressing Jurkat cells were treated with DOX (5 μg/ml) for 24, 48, and 72 h; whole-cell extracts were prepared at different times after treatment with DOX and were incubated with fluorogenic substrates for caspase 8 (■) and caspase 9 (▴) as described in Materials and Methods. Caspase activity is represented by the fluorescence ratio between DOX-induced versus noninduced cells.

    Journal: Journal of Virology

    Article Title: The IRF-3 Transcription Factor Mediates Sendai Virus-Induced Apoptosis

    doi:

    Figure Lengend Snippet: Caspase 8 and caspase 9 are involved in IRF-3-dependent activation of CPP-32/caspase-3. (A) rtTA-Jurkat IRF-3(5D) cells were cultured in the presence of 4 μg of APO-1-3 per ml or 50 μM Etoposide for 48 h in the continuous presence of caspase blockers zVAD, zIETH, and zLEHD (200 μM), as indicated. Viability was evaluated by using trypan blue exclusion. (B) wtIRF-3-expressing Jurkat cells were treated for 48 h with DOX (5 μg/ml) to stimulate IRF-3 production and were then infected with Sendai virus (400 HAU/ml/10 6 cells) for 24, 48, or 72 h in the continuous presence of caspase blockers zVAD, zIETH, and zLEHD (200 μM). Viability was evaluated by using trypan blue exclusion. (C) IRF-3(5D)-expressing Jurkat cells were treated with DOX (5 μg/ml) for 24, 48, or 72 h to stimulate IRF-3(5D) production. DOX treatment was accomplished in the continuous presence of caspase blockers zVAD, zIETH, and zLEHD (200 μM). Viability was evaluated by using trypan blue exclusion. Symbols in B and C: ■, Sendai virus; ▴, Sendai virus plus zLEHD; ⧫, Sendai virus plus zIETD; ●, Sendai virus plus zVAD. (D) IRF-3(5D)-expressing Jurkat cells were treated with DOX (5 μg/ml) for 24, 48, and 72 h; whole-cell extracts were prepared at different times after treatment with DOX and were incubated with fluorogenic substrates for caspase 8 (■) and caspase 9 (▴) as described in Materials and Methods. Caspase activity is represented by the fluorescence ratio between DOX-induced versus noninduced cells.

    Article Snippet: Nevertheless, some caspase 9 activity was observed and may be explained by recent data demonstrating that activated caspase 8 can recruit mitochondrial input into the caspase-9-mediated activation of caspase 3 via cleavage of Bid-2 ( ).

    Techniques: Activation Assay, Conditioned Place Preference, Cell Culture, Expressing, Infection, Incubation, Activity Assay, Fluorescence

    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: