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  • 95
    Millipore alpha ifn ifn α
    Regulation of CARD6 expression in BMDMs and splenocytes by IFNs and LPS. (A) Relative CARD6, RIP2, and PKR mRNA expression in BMDMs. BMDMs were stimulated for 3 h with 100 U ml −1 <t>IFN-α</t> or IFN-β, 50 ng ml −1 IFN-γ, or 100 ng ml −1 LPS or the indicated combinations of IFNs and LPS. RNA expression was measured by quantitative real-time PCR analysis. Results shown are mRNA levels relative to mRNA levels in unstimulated controls. (B) CARD6 mRNA induction by IFN-γ is rapid and transient. BMDMs were stimulated for the indicated times with 100 U ml −1 IFN-γ, IFN-α, or IFN-β. Relative mRNA expression was determined as for panel A. (C) Confirmation of CARD6 mRNA induction. CARD6 +/+ and CARD6 −/− BMDMs were left untreated or stimulated with IFN-γ for 3 h, and CARD6 RNA expression was detected by Northern blotting. (D) Upregulation of the ΔCARD6 protein variant in WT BMDMs. CARD6 +/+ and CARD6 −/− BMDMs were stimulated with IFN-γ for the indicated times, and extracts were subjected to Western blotting to detect CARD6, RIP2, and actin. (E) Relative CARD6 and PKR mRNA expression in splenocytes. Freshly isolated CARD6 +/+ splenocytes were stimulated with IFN-γ in the absence or presence of LPS for the indicated times, and relative mRNA expression was determined as for panel A. (F) CARD6 protein expression in splenocytes. Left panel, CARD6 +/+ splenocytes were incubated with IFN-γ in the absence or presence of LPS for the indicated times, and the expression of the (Δ)CARD6 and ΔCARD6S proteins was determined by Western blotting. Right panel, base levels of CARD6 protein expression were determined in unstimulated WT splenic T cells and B cells and in WT splenocytes depleted of T and B cells. Protein extracts from CARD6 −/− splenocytes are included as controls in both panels. (G) Proteolytic CARD6 degradation is not altered by a mutation in a putative caspase-1 cleavage site or by caspase-1 inhibition. 293T cells were transfected with empty vector or the indicated CARD6 expression constructs in the absence or presence of expression constructs for HA-tagged RIP2 or caspase-1. Where indicated, cells were also treated with caspase-1 inhibitor II (Calbiochem) at a final concentration of 10 μM. Cells were lysed at 36 h posttransfection, and expression levels of exogenous CARD6 (upper panel) or HA-tagged RIP2 and caspase-1 (lower panel) were analyzed using anti-moCARD6 antibody or anti-HA antibody, respectively. Results shown are from one trial, representative of at least three (A, B, D, and E) or two (F and G) independent experiments. *, cross-reactive bands.
    Alpha Ifn Ifn α, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Quanterix ifn α
    Regulation of CARD6 expression in BMDMs and splenocytes by IFNs and LPS. (A) Relative CARD6, RIP2, and PKR mRNA expression in BMDMs. BMDMs were stimulated for 3 h with 100 U ml −1 <t>IFN-α</t> or IFN-β, 50 ng ml −1 IFN-γ, or 100 ng ml −1 LPS or the indicated combinations of IFNs and LPS. RNA expression was measured by quantitative real-time PCR analysis. Results shown are mRNA levels relative to mRNA levels in unstimulated controls. (B) CARD6 mRNA induction by IFN-γ is rapid and transient. BMDMs were stimulated for the indicated times with 100 U ml −1 IFN-γ, IFN-α, or IFN-β. Relative mRNA expression was determined as for panel A. (C) Confirmation of CARD6 mRNA induction. CARD6 +/+ and CARD6 −/− BMDMs were left untreated or stimulated with IFN-γ for 3 h, and CARD6 RNA expression was detected by Northern blotting. (D) Upregulation of the ΔCARD6 protein variant in WT BMDMs. CARD6 +/+ and CARD6 −/− BMDMs were stimulated with IFN-γ for the indicated times, and extracts were subjected to Western blotting to detect CARD6, RIP2, and actin. (E) Relative CARD6 and PKR mRNA expression in splenocytes. Freshly isolated CARD6 +/+ splenocytes were stimulated with IFN-γ in the absence or presence of LPS for the indicated times, and relative mRNA expression was determined as for panel A. (F) CARD6 protein expression in splenocytes. Left panel, CARD6 +/+ splenocytes were incubated with IFN-γ in the absence or presence of LPS for the indicated times, and the expression of the (Δ)CARD6 and ΔCARD6S proteins was determined by Western blotting. Right panel, base levels of CARD6 protein expression were determined in unstimulated WT splenic T cells and B cells and in WT splenocytes depleted of T and B cells. Protein extracts from CARD6 −/− splenocytes are included as controls in both panels. (G) Proteolytic CARD6 degradation is not altered by a mutation in a putative caspase-1 cleavage site or by caspase-1 inhibition. 293T cells were transfected with empty vector or the indicated CARD6 expression constructs in the absence or presence of expression constructs for HA-tagged RIP2 or caspase-1. Where indicated, cells were also treated with caspase-1 inhibitor II (Calbiochem) at a final concentration of 10 μM. Cells were lysed at 36 h posttransfection, and expression levels of exogenous CARD6 (upper panel) or HA-tagged RIP2 and caspase-1 (lower panel) were analyzed using anti-moCARD6 antibody or anti-HA antibody, respectively. Results shown are from one trial, representative of at least three (A, B, D, and E) or two (F and G) independent experiments. *, cross-reactive bands.
    Ifn α, supplied by Quanterix, used in various techniques. Bioz Stars score: 94/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche ifn α
    Cell proliferative ability comparisons. The average colony-forming efficiency of Chang/HBx (A) was 29.3 ± 4.5, which was significantly different from that of Chang/pcDNA3.1 (B) (12.8 ± 2.6) and <t>IFN-α-Chang/HBx</t> (C) cells (13.5 ± 2.3) ( P
    Ifn α, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    PBL Assay ifn α
    Characterization of LCMV-induced long-lived antigen-specific CD8 T cells for STAT4 expression and in vivo responsiveness to <t>IFN-α.</t> To examine CD8 T cells in immune mice, LCMV-infected WT mice were maintained for ≥2 months after acute infection. Splenic leukocytes from uninfected mice, control vehicle-treated immune mice, and immune mice treated with 5 × 10 5 U of IFN-α administered i.v. at 90 min prior to harvest were prepared (A). The total CD8 + T cells and LCMV-Tet + and LCMV-Tet − CD8 T cell subsets were isolated. (B) Expression of total STAT4 and type 1 IFN induction of pSTAT4 were examined by Western blot and flow cytometric analyses. Total cells from uninfected mice and LCMV-immune mice that had been treated with the vehicle control (PBS) or CD8 and LCMV-Tet + and LCMV-Tet − CD8 T cell subsets from mice treated with IFN-α were prepared by FACS sorting. Extracted proteins were analyzed for STAT4, pSTAT4, and β-actin by Western blot analysis. Determination of cytoplasmic STAT4 and pSTAT4 within LCMV-specific CD8 T cells (Tet + compared to Tet − ) were evaluated by staining and flow cytometry. Gray areas in histograms represent results obtained with cells from control-treated immune mice, and the thick black lines represent results obtained with cells from IFN-α-treated immune mice. Results are representative of two experiments.
    Ifn α, supplied by PBL Assay, used in various techniques. Bioz Stars score: 91/100, based on 1090 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schering-Plough ifn α
    A , Addition of <t>IFN-α</t> amplifies TLR7-stimulated proliferation and differentiation of CD27 + and CD27 − B cells. Isolated CD27 + and CD27 − CVID or control B cells were stimulated with 500 μmol/L loxoribine in the presence or
    Ifn α, supplied by Schering-Plough, used in various techniques. Bioz Stars score: 93/100, based on 287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec ifn α
    a Increased STAT1 phosphorylation in patients’ monocytes after <t>IFN-α</t> and IFN-γ treatment. PBMC from patients (solid line) and healthy controls (dashed lines) were stimulated for 15 min with IFN-α (500 U/ml) or IFN-γ (100 ng/ml). Cells were fixed and permeabilized prior to staining with anti-CD14 and anti-phospho-STAT1 (pY701) antibodies. The gate was set on CD14+ monocytes. b Increased STAT1 phosphorylation in patients’ CD4+ T cells following IFN-α and IL-27 stimulation. PBMC were stimulated with either IFN-α (10,000 U/ml) or IL-27 (200 ng/ml) for 7.5, 15 and 30 min. Cells were then treated as described in ( a ) and phospho-STAT1 levels were evaluated gating on CD4+ T cells (Red – unstimulated patient cells; blue – stimulated healthy control cells; orange – stimulated patient cells)
    Ifn α, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 93/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    PBL Biomedical Laboratories ifn α
    pDC retain largely normal function in response to TLR7 stimulation during acute SIV infection. (A) Left: Contour plots demonstrating the gating strategy used to identify TNF-α + pDC in PBMC from SIV-naïve and SIV-infected monkeys following stimulation with 3M-007. Numbers represent the percentage of TNF-α + pDC within the indicated gates. Right: Percent of TNF-α + pDC in PBMC from SIV-naïve and SIV-infected monkeys in response to media or 3M-007. Bars represent the median and error bars the 95% confidence interval. (B) Left: Representative contour plots demonstrating the gating strategy used to identify TNF-α + , <t>IFN-α</t> + , or TNF-α + / IFN-α + pDC in lymph nodes following 3M-007 stimulation in the absence or presence of Brefeldin A (BFA). Numbers in parentheses represent the percentage of cells expressing the respective cytokines. Right: Percent of lymph node pDC from SIV-naïve and SIV-infected monkeys expressing TNF-α, IFN-α, or both in response to TLR7/8 agonist. Bars represent the median and error bars the 95% confidence interval. NS = not significant.
    Ifn α, supplied by PBL Biomedical Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 769 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioLegend ifn α
    A schematic of the mouse Batf2 gene, genotyping results, and the s.c. inoculated mouse tumor model. ( A ) A schematic of the mouse Batf2 gene, the targeting vector, and the targeted allele. ( B ) Genotyping PCR of Batf2 +/+ , Batf2 +/− , and Batf2 −/− mice. As indicated in A , primers shextra and wild together detect a fragment of the WT genomic locus, while primers shextra and PGKRC2 together detect a fragment of the mutant allele. ( C ) The relative expression levels of Batf2 mRNA in BMDMs from Batf2 −/− or WT littermates following stimulation by LPS (100 ng/mL) and <t>IFN-γ</t> (30 ng/mL) were quantified using qPCR ( Left ). The Batf2 mRNA levels in BMDMs from Batf2 −/− or WT littermates following stimulation with 10 3 U/mL of recombinant <t>IFN-α</t> were quantified using qPCR ( Right ). Error bars indicate ±SEM ( n = 2). ( D ) WT and Batf2 −/− mice were treated daily with imiquimod cream on shaved back skin for 6 d. The resulting erythema scores on days 4−6 are shown. Error bars indicate ±SEM ( n = 6). * P
    Ifn α, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam ifn α
    <t>IFN-α</t> protein expression was significantly increased in lung after MAB microparticle challenge. (A) Shows a representative image of western blot bands, (B) shows significant difference in IFN-α protein expression in the lung of challenged mice (sample size 4 for each group).
    Ifn α, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher ifn α
    Immunostimulatory activity tests of Lys3-modivariants. Human PBMCs were stimulated with DOTAP encapsulated RNA species at indicated concentrations. Splint-ligated mv#1 and mv#2 containing half part of native tRNA Lys 3 and unmodified RNAs as well as native tRNA Lys 3 were tested on three individual donors in duplicate wells. <t>IFN-α</t> levels were measured in cell-free supernatants 20 h post-transfection. To account for donor variation results were normalized to the amount of IFN-α induced by 0.5 μg/ml bRNA. Each bar illustrates mean +SEM.
    Ifn α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1077 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad ifn α
    NV RNA replication is sensitive to type I and III <t>IFN</t> treatment. (A) Effect of type I and III IFN pretreatment on NV RNA replication. 293FT cells were pretreated with increasing doses of type I (100 and 1,000 U/ml <t>IFN-α</t> or IFN-β) or III (10 and 100 ng/ml IL-29) IFN for 24 h and then transfected with carrier RNA (−) or NV RNA (+) and incubated for 48 h. NV VP1 in cell lysate was detected by Western blotting. Actin served as an equal loading control. (B) Effect of posttransfection IFN treatment on NV RNA replication. 293FT cells were transfected with NV RNA and treated with 1,000 U/ml IFN-β at 12 h post-RNA transfection. Cells were lysed at 48 h post-RNA transfection, and NV VP1 in cell lysate was detected by IP and Western blotting. (C) Immunofluorescence staining of NV VP1 in NV RNA-transfected 293FT cells either untreated (no IFN) or pretreated with 1,000 U/ml IFN-β. Cells were fixed at 48 h post-RNA transfection and stained with guinea pig antibody to NV VP1. Scale bars, 200 μm.
    Ifn α, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenScript ifn α
    Capture of SUMOylated PML and p53 using SUBE-l. NB4 cells (A) or MCF7 (B) were pre-treated with MG-132 before 1 h of ATO treatment. (C) SUMO and PML can be co-localized in the nuclear bodies (NB) of NB4 cells under the same conditions than (A or B). SUMOylated p53 can be captured by SUBE-l (D). HEK-293 were untreated or treated with <t>IFN-α</t> or infected with VSV. Input, flow-through (FT) and bound fractions were analysed with anti-SUMO-2/-3, PML or p53 antibodies. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as loading control.
    Ifn α, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bender MedSystems ifn α
    2'-O-methylated RNA inhibits <t>IFN-α,</t> TNF and IL-12p40 production with similar efficiency. a-c PBMCs were cotransfected overnight with 1 µg/ml bRNA purified from S. aureus and different ratios of 2'-O-methylated inhibitory RNA (inh. RNA). Levels of IFN-α ( a ), TNF ( b ) and IL-12p40 ( c ) were analysed in cell-free supernatants by ELISA. Data were normalized to cytokine production induced by bRNA alone to account for donor variation. Curve fit and IC 50 ( d ) were calculated with R software using the 4-parameter Weibull function. Each data point represents the average value of 2–6 independent experiments with different donors.
    Ifn α, supplied by Bender MedSystems, used in various techniques. Bioz Stars score: 92/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Accurate Chemical & Scientific Corporation ifn α
    2'-O-methylated RNA inhibits <t>IFN-α,</t> TNF and IL-12p40 production with similar efficiency. a-c PBMCs were cotransfected overnight with 1 µg/ml bRNA purified from S. aureus and different ratios of 2'-O-methylated inhibitory RNA (inh. RNA). Levels of IFN-α ( a ), TNF ( b ) and IL-12p40 ( c ) were analysed in cell-free supernatants by ELISA. Data were normalized to cytokine production induced by bRNA alone to account for donor variation. Curve fit and IC 50 ( d ) were calculated with R software using the 4-parameter Weibull function. Each data point represents the average value of 2–6 independent experiments with different donors.
    Ifn α, supplied by Accurate Chemical & Scientific Corporation, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co ifn α
    Comparison of protein microarray performance with an established bead-based assay. A , Corresponding MFI values from protein microarrays and a bead-based assay were plotted for the targets GM-CSF and <t>IFN-α,</t> and Pearson correlation coefficients
    Ifn α, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ifn α
    TRAIL induction is driven primarily by cytokines. (A) PBMC were infected with influenza. After infection, decreasing numbers of cells (10 6 – 1.25 × 10 5 cells) were aliquoted into 2 ml of media. After 24 h culture, <t>IFN-α</t> levels
    Ifn α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology ifn α
    Expression of <t>IFN-α</t> and TNF-α in A498 xenografts after rIL-22 treatment. Western blot assay was performed to detect the expression of IFN-α and TNF-α in the A498 cell xenografts. Neither the expression of IFN-α nor TNF-α were increased significantly in A498 cell xenografts treated with rIL-22 (P > 0.05 compared with control). N = 2.
    Ifn α, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    InvivoGen ifn α
    Activation of mouse-derived dendritic cells (DCs) by LNP-CpGs in vitro . Mouse-derived DCs were treated with CpG ODN or with each LNP-CpG for 24 h in vitro . The CpG ODN content was the same between the cells treated with CpG ODN alone and those treated with LNP-CpGs. (A) Cytokine production. Levels of IL-12 p40 and <t>IFN-α</t> in the supernatants were measured by ELISA. (B) Expression of co-stimulatory molecules. Expression of CD80 and CD86 on DCs was measured by flow cytometry; percentages of positive DCs are shown. (A,B) n = 5 per group. Data are means ± SD. † P
    Ifn α, supplied by InvivoGen, used in various techniques. Bioz Stars score: 93/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hycult Biotech ifn α
    <t>IFN-α-induced</t> Trex1 expression is IFNAR1-, STAT1-, and STAT2-dependent.
    Ifn α, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 92/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa ifn α
    Opn-dependent in vivo response to HSV-1 infection. ( a ) <t>IFN-α</t> concentrations from Opn wild-type (WT) and Opn-deficient (KO) pDC culture supernatants after incubation in vitro with ultraviolet irradiation–treated (UV-irradiated) HSV-1 for
    Ifn α, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Leinco Technologies ifn α
    Blockade of <t>IFN-α</t> significantly reduces islet immunopathology in Rip -LCMV mice infected with virus. Rip- LCMV mice were treated with IgG isotype control antibody (1 mg; Clone PIP), antibody to IFNAR (1 mg; Clone: MAR1-5A3), antibody to IFN-α
    Ifn α, supplied by Leinco Technologies, used in various techniques. Bioz Stars score: 91/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axxora ifn α
    Effect of IAV H3N2 NS1 protein mutations on innate immune responses induced by SeV infection. Human 293T cells were transiently transfected using the calcium phosphate method with the indicated pCAGGS NS1-expressing plasmids, together with plasmids expressing Fluc under the control of an <t>IFN-β</t> (A) or ISRE promoter (B). At 24 hpt, cells were mock infected (M) or infected with SeV (Cantell strain) (+SeV) to induce activation of the promoters, and 16 hpi cell lysates were prepared for reporter gene expression. (A and B) Reporter Firefly expression was measured by luminescence. Data represented show the means and standard deviations of the results determined for triplicate wells. Experiments were repeated 3 times in triplicate wells with similar results. P values determined using Student's t test are indicated. (C) At 16 h after SeV infection, TCS were collected and, after UV inactivation, were used to treat fresh A549 cells. Alternatively, A549 cells were treated with 2,500 U/ml of universal <t>IFN-α</t> as a control (picture on the right). After 24 h of incubation, cells were infected (MOI, 0.001) with the IFN-sensitive VSV-GFP. At 16 hpi, VSV-GFP-infected cells were observed under a fluorescence microscope. (D) At 16 h after SeV infection, cellular extracts were obtained and the expression of ISG15 and actin were evaluated by Western blotting, using antibodies specific to ISG15 and to actin (as a loading control). Western blots were quantified by densitometry using the software ImageJ (v1.46), and the amounts of ISG15 protein were normalized to the amounts of actin protein (numbers below the ISG15 blot; ND, not detected). Molecular mass markers (in kilodaltons) are indicated on the right. The squares represent the NS1 variants found in the subjects ( Table 4 ), showing the numbers of patients for each NS1 variant below the squares.
    Ifn α, supplied by Axxora, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Mab Technologies ifn α
    Resting levels of cellular proliferation and cell culture supernatant cytokines <t>IFN-γ,</t> IL-17, IL-10, <t>IFN-α,</t> and sCTLA-4 isolated from PBMC of SLE patients or age- and sex-matched healthy donors following incubation for 5 days ( n = 45 per group; * p
    Ifn α, supplied by Mab Technologies, used in various techniques. Bioz Stars score: 93/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen ifn α
    Specificity of <t>IFN-α</t> expressed by rhIFN-α BCG in regulating human peripheral blood mononuclear cell (PBMC) IFN-γ, IL-10 and IFN-inducible protein 10 (IP-10) production. PBMC were incubated with rhIFN-α BCG (rBCG, 0·01 OD 600 /ml) or rBCG (0·01 OD 600 /ml) plus neutralizing anti-IFN-α antibody (3 μg/ml) for 3 days. As controls, PBMC were incubated with medium, IFN-α (50 IU/ml), MV261 BCG (0·01 OD 600 /ml), or MV261 BCG (0·01 OD 600 /ml) plus IFN-α (50 IU/ml). IFN-γ, IL-10 and IP-10 in the cultures were quantified and the values were expressed as means ±s.d. from triplicate-well incubations.
    Ifn α, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Regulation of CARD6 expression in BMDMs and splenocytes by IFNs and LPS. (A) Relative CARD6, RIP2, and PKR mRNA expression in BMDMs. BMDMs were stimulated for 3 h with 100 U ml −1 IFN-α or IFN-β, 50 ng ml −1 IFN-γ, or 100 ng ml −1 LPS or the indicated combinations of IFNs and LPS. RNA expression was measured by quantitative real-time PCR analysis. Results shown are mRNA levels relative to mRNA levels in unstimulated controls. (B) CARD6 mRNA induction by IFN-γ is rapid and transient. BMDMs were stimulated for the indicated times with 100 U ml −1 IFN-γ, IFN-α, or IFN-β. Relative mRNA expression was determined as for panel A. (C) Confirmation of CARD6 mRNA induction. CARD6 +/+ and CARD6 −/− BMDMs were left untreated or stimulated with IFN-γ for 3 h, and CARD6 RNA expression was detected by Northern blotting. (D) Upregulation of the ΔCARD6 protein variant in WT BMDMs. CARD6 +/+ and CARD6 −/− BMDMs were stimulated with IFN-γ for the indicated times, and extracts were subjected to Western blotting to detect CARD6, RIP2, and actin. (E) Relative CARD6 and PKR mRNA expression in splenocytes. Freshly isolated CARD6 +/+ splenocytes were stimulated with IFN-γ in the absence or presence of LPS for the indicated times, and relative mRNA expression was determined as for panel A. (F) CARD6 protein expression in splenocytes. Left panel, CARD6 +/+ splenocytes were incubated with IFN-γ in the absence or presence of LPS for the indicated times, and the expression of the (Δ)CARD6 and ΔCARD6S proteins was determined by Western blotting. Right panel, base levels of CARD6 protein expression were determined in unstimulated WT splenic T cells and B cells and in WT splenocytes depleted of T and B cells. Protein extracts from CARD6 −/− splenocytes are included as controls in both panels. (G) Proteolytic CARD6 degradation is not altered by a mutation in a putative caspase-1 cleavage site or by caspase-1 inhibition. 293T cells were transfected with empty vector or the indicated CARD6 expression constructs in the absence or presence of expression constructs for HA-tagged RIP2 or caspase-1. Where indicated, cells were also treated with caspase-1 inhibitor II (Calbiochem) at a final concentration of 10 μM. Cells were lysed at 36 h posttransfection, and expression levels of exogenous CARD6 (upper panel) or HA-tagged RIP2 and caspase-1 (lower panel) were analyzed using anti-moCARD6 antibody or anti-HA antibody, respectively. Results shown are from one trial, representative of at least three (A, B, D, and E) or two (F and G) independent experiments. *, cross-reactive bands.

    Journal: Molecular and Cellular Biology

    Article Title: CARD6 Is Interferon Inducible but Not Involved in Nucleotide-Binding Oligomerization Domain Protein Signaling Leading to NF-?B Activation ▿

    doi: 10.1128/MCB.01359-07

    Figure Lengend Snippet: Regulation of CARD6 expression in BMDMs and splenocytes by IFNs and LPS. (A) Relative CARD6, RIP2, and PKR mRNA expression in BMDMs. BMDMs were stimulated for 3 h with 100 U ml −1 IFN-α or IFN-β, 50 ng ml −1 IFN-γ, or 100 ng ml −1 LPS or the indicated combinations of IFNs and LPS. RNA expression was measured by quantitative real-time PCR analysis. Results shown are mRNA levels relative to mRNA levels in unstimulated controls. (B) CARD6 mRNA induction by IFN-γ is rapid and transient. BMDMs were stimulated for the indicated times with 100 U ml −1 IFN-γ, IFN-α, or IFN-β. Relative mRNA expression was determined as for panel A. (C) Confirmation of CARD6 mRNA induction. CARD6 +/+ and CARD6 −/− BMDMs were left untreated or stimulated with IFN-γ for 3 h, and CARD6 RNA expression was detected by Northern blotting. (D) Upregulation of the ΔCARD6 protein variant in WT BMDMs. CARD6 +/+ and CARD6 −/− BMDMs were stimulated with IFN-γ for the indicated times, and extracts were subjected to Western blotting to detect CARD6, RIP2, and actin. (E) Relative CARD6 and PKR mRNA expression in splenocytes. Freshly isolated CARD6 +/+ splenocytes were stimulated with IFN-γ in the absence or presence of LPS for the indicated times, and relative mRNA expression was determined as for panel A. (F) CARD6 protein expression in splenocytes. Left panel, CARD6 +/+ splenocytes were incubated with IFN-γ in the absence or presence of LPS for the indicated times, and the expression of the (Δ)CARD6 and ΔCARD6S proteins was determined by Western blotting. Right panel, base levels of CARD6 protein expression were determined in unstimulated WT splenic T cells and B cells and in WT splenocytes depleted of T and B cells. Protein extracts from CARD6 −/− splenocytes are included as controls in both panels. (G) Proteolytic CARD6 degradation is not altered by a mutation in a putative caspase-1 cleavage site or by caspase-1 inhibition. 293T cells were transfected with empty vector or the indicated CARD6 expression constructs in the absence or presence of expression constructs for HA-tagged RIP2 or caspase-1. Where indicated, cells were also treated with caspase-1 inhibitor II (Calbiochem) at a final concentration of 10 μM. Cells were lysed at 36 h posttransfection, and expression levels of exogenous CARD6 (upper panel) or HA-tagged RIP2 and caspase-1 (lower panel) were analyzed using anti-moCARD6 antibody or anti-HA antibody, respectively. Results shown are from one trial, representative of at least three (A, B, D, and E) or two (F and G) independent experiments. *, cross-reactive bands.

    Article Snippet: If not indicated as “LPSp”, the LPS used in the stimulation experiments was from Sigma (L3012), as were alpha IFN (IFN-α) (100 U ml−1 ; Sigma) and beta IFN (IFN-β) (100 U ml−1 ; Sigma).

    Techniques: Expressing, RNA Expression, Real-time Polymerase Chain Reaction, Northern Blot, Variant Assay, Western Blot, Isolation, Incubation, Mutagenesis, Inhibition, Transfection, Plasmid Preparation, Construct, Concentration Assay

    Detection of DNA fragmentation. (A) The number of apoptotic MDA-MB-231 cells was quantified by flow cytometry using propidium iodide DNA staining after gene depletion under basal or TRAIL-treated conditions (i.e. measurement of the sub-G0/G1 peak in the fluorescence DNA histograms, right panels ). (B) Apoptosis was also evaluated in MDA-MB-231 by the inspection of DNA fragmentation by TUNEL (fluorescein-12-dUTP labeled fragmented DNA) staining ( right panels ). Cell nuclei were stained with Hoechst (blue fluorescence) to estimate the number of total cells. ( C ) A similar analysis was carried out in MCF-7 cells. The measurement of the sub-G0/G1 peaks ( right panels ) indicates a significantly higher number of apoptotic gene-depleted cells even under basal conditions ( left ). ( D ) TUNEL positive nuclei displaying green fluorescence are observed under basal conditions ( right ), although a higher number is clearly observed under TRAIL-induced conditions ( left ). siLUC was used as a negative control, XIAP was used as an anti-apoptotic reference in all experiments. EPSTI1 -depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. In apoptosis-induced conditions, cells were treated with TRAIL for 24h, at 80 or 100ng/mL respectively. Each bar represents the mean ±SD of three experiments performed in duplicate (* P

    Journal: PLoS ONE

    Article Title: Breast Cancer Genes PSMC3IP and EPSTI1 Play a Role in Apoptosis Regulation

    doi: 10.1371/journal.pone.0115352

    Figure Lengend Snippet: Detection of DNA fragmentation. (A) The number of apoptotic MDA-MB-231 cells was quantified by flow cytometry using propidium iodide DNA staining after gene depletion under basal or TRAIL-treated conditions (i.e. measurement of the sub-G0/G1 peak in the fluorescence DNA histograms, right panels ). (B) Apoptosis was also evaluated in MDA-MB-231 by the inspection of DNA fragmentation by TUNEL (fluorescein-12-dUTP labeled fragmented DNA) staining ( right panels ). Cell nuclei were stained with Hoechst (blue fluorescence) to estimate the number of total cells. ( C ) A similar analysis was carried out in MCF-7 cells. The measurement of the sub-G0/G1 peaks ( right panels ) indicates a significantly higher number of apoptotic gene-depleted cells even under basal conditions ( left ). ( D ) TUNEL positive nuclei displaying green fluorescence are observed under basal conditions ( right ), although a higher number is clearly observed under TRAIL-induced conditions ( left ). siLUC was used as a negative control, XIAP was used as an anti-apoptotic reference in all experiments. EPSTI1 -depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. In apoptosis-induced conditions, cells were treated with TRAIL for 24h, at 80 or 100ng/mL respectively. Each bar represents the mean ±SD of three experiments performed in duplicate (* P

    Article Snippet: For RNA interference-mediated gene silencing, cells were seeded and exposed to 50 nM of either gene-specific siRNA or non-targeting control siRNA (siLUC), using Lipofectamine RNAiMAX transfection reagent (Life Technologies) for 48 h. For EPSTI1 silencing, IFN-α (Chemicon, Millipore) was added to a final concentration of 1000 U/ml 8 hours before harvesting the cells.

    Techniques: Multiple Displacement Amplification, Flow Cytometry, Cytometry, Staining, Fluorescence, TUNEL Assay, Labeling, Negative Control

    Caspase-8 activity modulation. Caspase-8 activity was quantified by measuring the chromophore levels released from caspase-8 cleaved substrates. Overexpression of PSMC3IP or EPSTI1 in TRAIL-treated MDA-MB-231 (A) and MCF-7 cells (B) decrease caspase-8 activity based on the MYC-tag empty transfection vector (Vector) as control. Caspase-8 activity was also measured after gene silencing in MDA-MB-231 (C) and MCF-7 (D) cells, under basal or TRAIL-treated conditions. Genes were silenced using specific siRNAs targeting XIAP , PSMC3IP or EPSTI1 and siRNA against luciferase expression (siLUC) was used as a negative control. EPSTI1 -depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. MDA-MB-231 and MCF-7 cells were treated with TRAIL for 24h at 80 or 100ng/mL, respectively. XIAP was used as an anti-apoptotic reference in all experiments. Each bar represents the mean ±SD of three experiments performed in duplicate (* P

    Journal: PLoS ONE

    Article Title: Breast Cancer Genes PSMC3IP and EPSTI1 Play a Role in Apoptosis Regulation

    doi: 10.1371/journal.pone.0115352

    Figure Lengend Snippet: Caspase-8 activity modulation. Caspase-8 activity was quantified by measuring the chromophore levels released from caspase-8 cleaved substrates. Overexpression of PSMC3IP or EPSTI1 in TRAIL-treated MDA-MB-231 (A) and MCF-7 cells (B) decrease caspase-8 activity based on the MYC-tag empty transfection vector (Vector) as control. Caspase-8 activity was also measured after gene silencing in MDA-MB-231 (C) and MCF-7 (D) cells, under basal or TRAIL-treated conditions. Genes were silenced using specific siRNAs targeting XIAP , PSMC3IP or EPSTI1 and siRNA against luciferase expression (siLUC) was used as a negative control. EPSTI1 -depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. MDA-MB-231 and MCF-7 cells were treated with TRAIL for 24h at 80 or 100ng/mL, respectively. XIAP was used as an anti-apoptotic reference in all experiments. Each bar represents the mean ±SD of three experiments performed in duplicate (* P

    Article Snippet: For RNA interference-mediated gene silencing, cells were seeded and exposed to 50 nM of either gene-specific siRNA or non-targeting control siRNA (siLUC), using Lipofectamine RNAiMAX transfection reagent (Life Technologies) for 48 h. For EPSTI1 silencing, IFN-α (Chemicon, Millipore) was added to a final concentration of 1000 U/ml 8 hours before harvesting the cells.

    Techniques: Activity Assay, Over Expression, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Luciferase, Expressing, Negative Control

    Modulation of PSMC3IP and EPSTI1 expression in breast cancer cells. (A-B) Genes were overexpressed as Myc-tagged fusion proteins in different cell lines and protein relative levels were analysed based on MYC-tag empty transfection vector (Vector). (C-D) Endogenous gene expression was silenced using specific siRNA and depletion levels were analysed based on siRNA against luciferase expression (siLUC) as a negative control. Prior to depletion experiments, EPSTI1 expression was induced by treating cells with IFN-α at 1000 U/ml for 8h. XIAP was used as a reference anti-apoptotic protein in all experiments. Each bar represents the mean ±SD of three experiments performed in duplicate (* P

    Journal: PLoS ONE

    Article Title: Breast Cancer Genes PSMC3IP and EPSTI1 Play a Role in Apoptosis Regulation

    doi: 10.1371/journal.pone.0115352

    Figure Lengend Snippet: Modulation of PSMC3IP and EPSTI1 expression in breast cancer cells. (A-B) Genes were overexpressed as Myc-tagged fusion proteins in different cell lines and protein relative levels were analysed based on MYC-tag empty transfection vector (Vector). (C-D) Endogenous gene expression was silenced using specific siRNA and depletion levels were analysed based on siRNA against luciferase expression (siLUC) as a negative control. Prior to depletion experiments, EPSTI1 expression was induced by treating cells with IFN-α at 1000 U/ml for 8h. XIAP was used as a reference anti-apoptotic protein in all experiments. Each bar represents the mean ±SD of three experiments performed in duplicate (* P

    Article Snippet: For RNA interference-mediated gene silencing, cells were seeded and exposed to 50 nM of either gene-specific siRNA or non-targeting control siRNA (siLUC), using Lipofectamine RNAiMAX transfection reagent (Life Technologies) for 48 h. For EPSTI1 silencing, IFN-α (Chemicon, Millipore) was added to a final concentration of 1000 U/ml 8 hours before harvesting the cells.

    Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Negative Control

    Cell viability and recovery. Cell viability was determined by MTT absorbance assays (A) Histograms showing the viability of PSMC3IP or EPSTI1-overexpressing MDA-MB-231 cells and (B) MCF-7 cells under TRAIL-induced conditions. Based on empty vector (MYC-tag) as a negative control, we do not observe a significant recovery of apoptosis-induced cells after gene overexpression. (C) Viability measurement of gene-depleted MDA-MB-231 cells reveals that EPSTI1 depletion reduces about 50% of viability as compared to siLUC negative control. (D) Intriguingly, in MCF-7 cells, both PSMC3IP and EPSTI1 depletion lead to a decreased viability under basal but not under TRAIL-treated conditions. XIAP was used as an anti-apoptotic reference in all experiments. EPSTI1 -depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. In apoptosis-induced conditions, cells were treated with TRAIL for 24h, at 80 or 100ng/mL respectively. Each bar represents the mean ±SD of three experiments performed in duplicate (* P

    Journal: PLoS ONE

    Article Title: Breast Cancer Genes PSMC3IP and EPSTI1 Play a Role in Apoptosis Regulation

    doi: 10.1371/journal.pone.0115352

    Figure Lengend Snippet: Cell viability and recovery. Cell viability was determined by MTT absorbance assays (A) Histograms showing the viability of PSMC3IP or EPSTI1-overexpressing MDA-MB-231 cells and (B) MCF-7 cells under TRAIL-induced conditions. Based on empty vector (MYC-tag) as a negative control, we do not observe a significant recovery of apoptosis-induced cells after gene overexpression. (C) Viability measurement of gene-depleted MDA-MB-231 cells reveals that EPSTI1 depletion reduces about 50% of viability as compared to siLUC negative control. (D) Intriguingly, in MCF-7 cells, both PSMC3IP and EPSTI1 depletion lead to a decreased viability under basal but not under TRAIL-treated conditions. XIAP was used as an anti-apoptotic reference in all experiments. EPSTI1 -depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. In apoptosis-induced conditions, cells were treated with TRAIL for 24h, at 80 or 100ng/mL respectively. Each bar represents the mean ±SD of three experiments performed in duplicate (* P

    Article Snippet: For RNA interference-mediated gene silencing, cells were seeded and exposed to 50 nM of either gene-specific siRNA or non-targeting control siRNA (siLUC), using Lipofectamine RNAiMAX transfection reagent (Life Technologies) for 48 h. For EPSTI1 silencing, IFN-α (Chemicon, Millipore) was added to a final concentration of 1000 U/ml 8 hours before harvesting the cells.

    Techniques: MTT Assay, Multiple Displacement Amplification, Plasmid Preparation, Negative Control, Over Expression

    Caspase-3 activity modulation and analysis of cleaved PARP protein levels. (A) Caspase-3 activity was measured by colorimetric quantification of fluorescent products released from caspase-3 cleaved substrates.in TRAIL-treated MDA-MB-231 cells overexpressing XIAP, PSMC3IP or EPSTI1. None of the overexpressed genes was able to significantly decrease the activity relative to MYC-tag empty transfection vector (Vector) as control. (B) Caspase-3 activity was also measured in MDA-MB-231 cells under basal or TRAIL-treated conditions after gene silencing using specific siRNA targeting XIAP , PSMC3IP or EPSTI1 . (C) Immunoblot analysis of cleaved PARP protein levels in gene-overexpressing MDA-MB-231 cells under TRAIL conditions, using MYC-tag transfection vector (Vector) as a negative control, reveals an attenuation of downstream apoptotic cascades. (D) This effect is much more pronounced in TRAIL-treated MCF-7 cells. (E) Analysis of cleaved PARP protein levels after gene silencing in MDA-MB-231 cells. siLUC was used as a negative control. (F) The same analysis in MCF-7 cells shows a more pronounced effect, even under basal conditions. EPSTI1 -depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. In apoptosis-induced conditions, MDA-MB-231 and MCF-7 cells were treated with TRAIL for 24h at 80 or 100ng/mL, respectively. XIAP was used as an anti-apoptotic reference in all experiments. Each bar represents the mean ±SD of three experiments performed in duplicate (* P

    Journal: PLoS ONE

    Article Title: Breast Cancer Genes PSMC3IP and EPSTI1 Play a Role in Apoptosis Regulation

    doi: 10.1371/journal.pone.0115352

    Figure Lengend Snippet: Caspase-3 activity modulation and analysis of cleaved PARP protein levels. (A) Caspase-3 activity was measured by colorimetric quantification of fluorescent products released from caspase-3 cleaved substrates.in TRAIL-treated MDA-MB-231 cells overexpressing XIAP, PSMC3IP or EPSTI1. None of the overexpressed genes was able to significantly decrease the activity relative to MYC-tag empty transfection vector (Vector) as control. (B) Caspase-3 activity was also measured in MDA-MB-231 cells under basal or TRAIL-treated conditions after gene silencing using specific siRNA targeting XIAP , PSMC3IP or EPSTI1 . (C) Immunoblot analysis of cleaved PARP protein levels in gene-overexpressing MDA-MB-231 cells under TRAIL conditions, using MYC-tag transfection vector (Vector) as a negative control, reveals an attenuation of downstream apoptotic cascades. (D) This effect is much more pronounced in TRAIL-treated MCF-7 cells. (E) Analysis of cleaved PARP protein levels after gene silencing in MDA-MB-231 cells. siLUC was used as a negative control. (F) The same analysis in MCF-7 cells shows a more pronounced effect, even under basal conditions. EPSTI1 -depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. In apoptosis-induced conditions, MDA-MB-231 and MCF-7 cells were treated with TRAIL for 24h at 80 or 100ng/mL, respectively. XIAP was used as an anti-apoptotic reference in all experiments. Each bar represents the mean ±SD of three experiments performed in duplicate (* P

    Article Snippet: For RNA interference-mediated gene silencing, cells were seeded and exposed to 50 nM of either gene-specific siRNA or non-targeting control siRNA (siLUC), using Lipofectamine RNAiMAX transfection reagent (Life Technologies) for 48 h. For EPSTI1 silencing, IFN-α (Chemicon, Millipore) was added to a final concentration of 1000 U/ml 8 hours before harvesting the cells.

    Techniques: Activity Assay, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Negative Control

    Cell proliferative ability comparisons. The average colony-forming efficiency of Chang/HBx (A) was 29.3 ± 4.5, which was significantly different from that of Chang/pcDNA3.1 (B) (12.8 ± 2.6) and IFN-α-Chang/HBx (C) cells (13.5 ± 2.3) ( P

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Interferon-alpha restrains growth and invasive potential of hepatocellular carcinoma induced by hepatitis B virus X protein

    doi: 10.3748/wjg.14.5564

    Figure Lengend Snippet: Cell proliferative ability comparisons. The average colony-forming efficiency of Chang/HBx (A) was 29.3 ± 4.5, which was significantly different from that of Chang/pcDNA3.1 (B) (12.8 ± 2.6) and IFN-α-Chang/HBx (C) cells (13.5 ± 2.3) ( P

    Article Snippet: The IFN-α was purchased from Roche Pharmaceuticals Ltd. (Guangxi, China).

    Techniques:

    A: A nude mouse migration model with pcDNA3.1-HBx Chang cells, IFN-α-Chang/HBx cells and Chang/pcDNA3.1 cells were constructed on the 7th wk; B: The volume of neoplasms in the three groups were observed. The sizes of the hepatomas from Chang/pcDNA3.1 and IFN-α-Chang/HBx injected nude mice were obviously larger than the tumors of Chang/HBx injected mice; C: Neoplasm growth in the Chang/HBx-inoculated nude mice compared between IFN-α-Chang/HBx and the control Chang/pcDNA3.1-inoculated mice.

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Interferon-alpha restrains growth and invasive potential of hepatocellular carcinoma induced by hepatitis B virus X protein

    doi: 10.3748/wjg.14.5564

    Figure Lengend Snippet: A: A nude mouse migration model with pcDNA3.1-HBx Chang cells, IFN-α-Chang/HBx cells and Chang/pcDNA3.1 cells were constructed on the 7th wk; B: The volume of neoplasms in the three groups were observed. The sizes of the hepatomas from Chang/pcDNA3.1 and IFN-α-Chang/HBx injected nude mice were obviously larger than the tumors of Chang/HBx injected mice; C: Neoplasm growth in the Chang/HBx-inoculated nude mice compared between IFN-α-Chang/HBx and the control Chang/pcDNA3.1-inoculated mice.

    Article Snippet: The IFN-α was purchased from Roche Pharmaceuticals Ltd. (Guangxi, China).

    Techniques: Migration, Construct, Injection, Mouse Assay

    The growth curve of Chang/HBx cells shows a shift to the left from that of Chang/pcDNA3.1 and IFN-α-Chang/HBx cells.

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Interferon-alpha restrains growth and invasive potential of hepatocellular carcinoma induced by hepatitis B virus X protein

    doi: 10.3748/wjg.14.5564

    Figure Lengend Snippet: The growth curve of Chang/HBx cells shows a shift to the left from that of Chang/pcDNA3.1 and IFN-α-Chang/HBx cells.

    Article Snippet: The IFN-α was purchased from Roche Pharmaceuticals Ltd. (Guangxi, China).

    Techniques:

    HBx promotes the invasive capacity of Chang liver cells in vitro . Wound-healing assays were performed at 0 and 24 h and observed under a phase-contrast microscope (× 100). A: Chang liver cells with pcDNA3.1-HBx were disrupted, and wound healing occurred after 24 h; B: IFN-α-Chang/HBx cells were disrupted, and the wound did not heal completely after 24 h; C: Chang cells with pcDNA3.1 were disrupted, and the wound did not heal completely after 24 h.

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Interferon-alpha restrains growth and invasive potential of hepatocellular carcinoma induced by hepatitis B virus X protein

    doi: 10.3748/wjg.14.5564

    Figure Lengend Snippet: HBx promotes the invasive capacity of Chang liver cells in vitro . Wound-healing assays were performed at 0 and 24 h and observed under a phase-contrast microscope (× 100). A: Chang liver cells with pcDNA3.1-HBx were disrupted, and wound healing occurred after 24 h; B: IFN-α-Chang/HBx cells were disrupted, and the wound did not heal completely after 24 h; C: Chang cells with pcDNA3.1 were disrupted, and the wound did not heal completely after 24 h.

    Article Snippet: The IFN-α was purchased from Roche Pharmaceuticals Ltd. (Guangxi, China).

    Techniques: In Vitro, Microscopy

    HBx promotes the invasive capacity of Chang liver cells in vitro . A: Chang cells transfected with pcDNA3.1-HBx invaded through the Matrigel were counted on the underside of the transwell filter and compared with Chang/HBx cells (B) and control cells Chang/pcDNA3.1 (C) (× 200). There was no difference between IFN-α-Chang/HBx cells and Chang/pcDNA3.1; D: The average number of migrated cells per site seen under a high-power microscope (× 400) was 41.6 ± 3.1 for the transfected Chang/HBx cells and 7.4 ± 1.2 for IFN-α-Chang/HBx cells and 9.2 ± 1.6 for the control Chang/pcDNA3.1 cells.

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Interferon-alpha restrains growth and invasive potential of hepatocellular carcinoma induced by hepatitis B virus X protein

    doi: 10.3748/wjg.14.5564

    Figure Lengend Snippet: HBx promotes the invasive capacity of Chang liver cells in vitro . A: Chang cells transfected with pcDNA3.1-HBx invaded through the Matrigel were counted on the underside of the transwell filter and compared with Chang/HBx cells (B) and control cells Chang/pcDNA3.1 (C) (× 200). There was no difference between IFN-α-Chang/HBx cells and Chang/pcDNA3.1; D: The average number of migrated cells per site seen under a high-power microscope (× 400) was 41.6 ± 3.1 for the transfected Chang/HBx cells and 7.4 ± 1.2 for IFN-α-Chang/HBx cells and 9.2 ± 1.6 for the control Chang/pcDNA3.1 cells.

    Article Snippet: The IFN-α was purchased from Roche Pharmaceuticals Ltd. (Guangxi, China).

    Techniques: In Vitro, Transfection, Microscopy

    Characterization of LCMV-induced long-lived antigen-specific CD8 T cells for STAT4 expression and in vivo responsiveness to IFN-α. To examine CD8 T cells in immune mice, LCMV-infected WT mice were maintained for ≥2 months after acute infection. Splenic leukocytes from uninfected mice, control vehicle-treated immune mice, and immune mice treated with 5 × 10 5 U of IFN-α administered i.v. at 90 min prior to harvest were prepared (A). The total CD8 + T cells and LCMV-Tet + and LCMV-Tet − CD8 T cell subsets were isolated. (B) Expression of total STAT4 and type 1 IFN induction of pSTAT4 were examined by Western blot and flow cytometric analyses. Total cells from uninfected mice and LCMV-immune mice that had been treated with the vehicle control (PBS) or CD8 and LCMV-Tet + and LCMV-Tet − CD8 T cell subsets from mice treated with IFN-α were prepared by FACS sorting. Extracted proteins were analyzed for STAT4, pSTAT4, and β-actin by Western blot analysis. Determination of cytoplasmic STAT4 and pSTAT4 within LCMV-specific CD8 T cells (Tet + compared to Tet − ) were evaluated by staining and flow cytometry. Gray areas in histograms represent results obtained with cells from control-treated immune mice, and the thick black lines represent results obtained with cells from IFN-α-treated immune mice. Results are representative of two experiments.

    Journal: mBio

    Article Title: CD8 T Cells in Innate Immune Responses: Using STAT4-Dependent but Antigen-Independent Pathways to Gamma Interferon during Viral Infection

    doi: 10.1128/mBio.01978-14

    Figure Lengend Snippet: Characterization of LCMV-induced long-lived antigen-specific CD8 T cells for STAT4 expression and in vivo responsiveness to IFN-α. To examine CD8 T cells in immune mice, LCMV-infected WT mice were maintained for ≥2 months after acute infection. Splenic leukocytes from uninfected mice, control vehicle-treated immune mice, and immune mice treated with 5 × 10 5 U of IFN-α administered i.v. at 90 min prior to harvest were prepared (A). The total CD8 + T cells and LCMV-Tet + and LCMV-Tet − CD8 T cell subsets were isolated. (B) Expression of total STAT4 and type 1 IFN induction of pSTAT4 were examined by Western blot and flow cytometric analyses. Total cells from uninfected mice and LCMV-immune mice that had been treated with the vehicle control (PBS) or CD8 and LCMV-Tet + and LCMV-Tet − CD8 T cell subsets from mice treated with IFN-α were prepared by FACS sorting. Extracted proteins were analyzed for STAT4, pSTAT4, and β-actin by Western blot analysis. Determination of cytoplasmic STAT4 and pSTAT4 within LCMV-specific CD8 T cells (Tet + compared to Tet − ) were evaluated by staining and flow cytometry. Gray areas in histograms represent results obtained with cells from control-treated immune mice, and the thick black lines represent results obtained with cells from IFN-α-treated immune mice. Results are representative of two experiments.

    Article Snippet: For in vivo IFN-α treatment, LCMV-immune mice were i.v. injected with 5 × 105 U of IFN-α (PBL Interferon Source) at 90 min before harvest.

    Techniques: Expressing, In Vivo, Mouse Assay, Infection, Isolation, Western Blot, Flow Cytometry, FACS, Staining, Cytometry

    Innate cytokine responses during secondary infections of LCMV-immune mice with LCMVcl13. Serum samples were collected from immune mice and from immune mice after a secondary i.v. infection with 4 × 10 6 PFU of LCMV clone 13 (LCMVc13) for 8 h, 16 h, 1 day, 1.5 days, 2.5 days, and 3.5 days as indicated (A). Antiviral biological activity was determined in a viral protection assay (B). Circulating levels of IFN-α and IFN-β (C) and IL-18 (D) were measured by ELISA, and those of IL-12 and IFN-γ (D) were measured by CBA as described in Materials and Methods. The samples were collected from eight independent experiments. Symbols represent results from individual mice with three to nine samples from multiple experiments used for each time point. Means ± SEMs (error bars) are shown. Means are connected by solid lines. The limit of detection by the assay is indicated by the dashed line.

    Journal: mBio

    Article Title: CD8 T Cells in Innate Immune Responses: Using STAT4-Dependent but Antigen-Independent Pathways to Gamma Interferon during Viral Infection

    doi: 10.1128/mBio.01978-14

    Figure Lengend Snippet: Innate cytokine responses during secondary infections of LCMV-immune mice with LCMVcl13. Serum samples were collected from immune mice and from immune mice after a secondary i.v. infection with 4 × 10 6 PFU of LCMV clone 13 (LCMVc13) for 8 h, 16 h, 1 day, 1.5 days, 2.5 days, and 3.5 days as indicated (A). Antiviral biological activity was determined in a viral protection assay (B). Circulating levels of IFN-α and IFN-β (C) and IL-18 (D) were measured by ELISA, and those of IL-12 and IFN-γ (D) were measured by CBA as described in Materials and Methods. The samples were collected from eight independent experiments. Symbols represent results from individual mice with three to nine samples from multiple experiments used for each time point. Means ± SEMs (error bars) are shown. Means are connected by solid lines. The limit of detection by the assay is indicated by the dashed line.

    Article Snippet: For in vivo IFN-α treatment, LCMV-immune mice were i.v. injected with 5 × 105 U of IFN-α (PBL Interferon Source) at 90 min before harvest.

    Techniques: Mouse Assay, Infection, Activity Assay, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay

    Innate cytokine responses in serum and NK cells during primary MCMV infections of LCMV-immune mice. Serum and splenic leukocyte samples were collected from LCMV-immune (≥2 mpi) mice and from LCMV-immune mice after a primary i.p. infection with 10 to 15,000 PFU MCMV at 8 h, 16 h, 1 day, 1.5 days, and 3.5 days before harvest as indicated (A). Circulating levels of IFN-α and IFN-β (B) and IL-18 (C) were measured by ELISA, and those of IL-12 and IFN-γ (C) were measured by CBA, as described in Materials and Methods. The samples were collected from three independent experiments. The means are connected by solid lines. The limit of detection by the assays is indicated by the dashed line. (D) Splenic leukocytes were analyzed for NK1.1 + TCRβ − NK cell populations expressing intracellular IFN-γ. The IFN-γ proportions and yields of NK1.1 + TCRβ − NK cells were determined after 4 h of ex vivo incubation in brefeldin without further stimulation. Representative staining of single samples is shown as plots (D) with histograms of IFN-γ staining within NK cells from uninfected LCMV-immune (≥2 mpi) (blue line) or day 1.5 MCMV-challenged (red line) LCMV-immune mice presented. The percentages (F) and yields (G) of NK cells expressing IFN-γ are shown. Symbols represent results from individual mice with three to nine samples from multiple experiments (B, C) or three mice per group in one experiment (D to G) used for each time point shown. Means ± SEMs (error bars) are shown. Mean NK cell percentages and IFN-γ expression values are connected by solid lines.

    Journal: mBio

    Article Title: CD8 T Cells in Innate Immune Responses: Using STAT4-Dependent but Antigen-Independent Pathways to Gamma Interferon during Viral Infection

    doi: 10.1128/mBio.01978-14

    Figure Lengend Snippet: Innate cytokine responses in serum and NK cells during primary MCMV infections of LCMV-immune mice. Serum and splenic leukocyte samples were collected from LCMV-immune (≥2 mpi) mice and from LCMV-immune mice after a primary i.p. infection with 10 to 15,000 PFU MCMV at 8 h, 16 h, 1 day, 1.5 days, and 3.5 days before harvest as indicated (A). Circulating levels of IFN-α and IFN-β (B) and IL-18 (C) were measured by ELISA, and those of IL-12 and IFN-γ (C) were measured by CBA, as described in Materials and Methods. The samples were collected from three independent experiments. The means are connected by solid lines. The limit of detection by the assays is indicated by the dashed line. (D) Splenic leukocytes were analyzed for NK1.1 + TCRβ − NK cell populations expressing intracellular IFN-γ. The IFN-γ proportions and yields of NK1.1 + TCRβ − NK cells were determined after 4 h of ex vivo incubation in brefeldin without further stimulation. Representative staining of single samples is shown as plots (D) with histograms of IFN-γ staining within NK cells from uninfected LCMV-immune (≥2 mpi) (blue line) or day 1.5 MCMV-challenged (red line) LCMV-immune mice presented. The percentages (F) and yields (G) of NK cells expressing IFN-γ are shown. Symbols represent results from individual mice with three to nine samples from multiple experiments (B, C) or three mice per group in one experiment (D to G) used for each time point shown. Means ± SEMs (error bars) are shown. Mean NK cell percentages and IFN-γ expression values are connected by solid lines.

    Article Snippet: For in vivo IFN-α treatment, LCMV-immune mice were i.v. injected with 5 × 105 U of IFN-α (PBL Interferon Source) at 90 min before harvest.

    Techniques: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay, Expressing, Ex Vivo, Incubation, Staining

    Changing responses to stimulation in CD8 T cells during acute LCMV infection. WT (A to C) or WT and STAT4 −/− (STAT4 − ) (D to F) B6 mice were left uninfected (day 0) or i.p. infected with 1 × 10 5 PFU LCMVclE350 for 8 days. (A, D) Flow cytometric analyses were used to identify CD8 + TCRβ + cells within splenic leukocytes and further analyzed as LCMV specific (LCMV-Tet + ) or nonspecific (LCMV-Tet − ) on the basis of binding of pools of MHC class 1 tetramers presenting the three known immunodominant LCMV epitopes in the H-2 b background, i.e., NP396-404 (NP396), GP276-286 (GP276), and GP33-41 (GP33). Responses to cytokine or TCR stimulation ex vivo were examined as indicated after 24 h in culture under control conditions (medium), after treatment with IFN-α at 1,000 U/ml, IL-12 at 10 ng/ml, or IL-18 at 10 ng/ml alone and combinations of IFN-α and IL-18 (B, C, E), with plate-bound antibodies against CD3 (αCD3ε) (E, F), and with pooled immunodominant LCMV peptides, i.e., NP396, GP276, and GP33 (F). (B, E, F) Induction of IFN-γ (green) was evaluated by intracellular staining, and that of CD25 (red) was evaluated by cell surface staining. Gray-shaded histograms are results of staining under control conditions. (C) Responses of day 8 LCMV-Tet + (color-shaded histograms) compared to LCMV-Tet − (gray-shaded histograms) CD8 T cells are shown. Histograms are results from an individual mouse. Values are mean results ± SEMs obtained with four mice per group in one experiment. The experiments were repeated twice. Significant differences between the responses of WT and STAT4 − groups are indicated as follows: ***, P ≤ 0.0001; **, P ≤ 0.005.

    Journal: mBio

    Article Title: CD8 T Cells in Innate Immune Responses: Using STAT4-Dependent but Antigen-Independent Pathways to Gamma Interferon during Viral Infection

    doi: 10.1128/mBio.01978-14

    Figure Lengend Snippet: Changing responses to stimulation in CD8 T cells during acute LCMV infection. WT (A to C) or WT and STAT4 −/− (STAT4 − ) (D to F) B6 mice were left uninfected (day 0) or i.p. infected with 1 × 10 5 PFU LCMVclE350 for 8 days. (A, D) Flow cytometric analyses were used to identify CD8 + TCRβ + cells within splenic leukocytes and further analyzed as LCMV specific (LCMV-Tet + ) or nonspecific (LCMV-Tet − ) on the basis of binding of pools of MHC class 1 tetramers presenting the three known immunodominant LCMV epitopes in the H-2 b background, i.e., NP396-404 (NP396), GP276-286 (GP276), and GP33-41 (GP33). Responses to cytokine or TCR stimulation ex vivo were examined as indicated after 24 h in culture under control conditions (medium), after treatment with IFN-α at 1,000 U/ml, IL-12 at 10 ng/ml, or IL-18 at 10 ng/ml alone and combinations of IFN-α and IL-18 (B, C, E), with plate-bound antibodies against CD3 (αCD3ε) (E, F), and with pooled immunodominant LCMV peptides, i.e., NP396, GP276, and GP33 (F). (B, E, F) Induction of IFN-γ (green) was evaluated by intracellular staining, and that of CD25 (red) was evaluated by cell surface staining. Gray-shaded histograms are results of staining under control conditions. (C) Responses of day 8 LCMV-Tet + (color-shaded histograms) compared to LCMV-Tet − (gray-shaded histograms) CD8 T cells are shown. Histograms are results from an individual mouse. Values are mean results ± SEMs obtained with four mice per group in one experiment. The experiments were repeated twice. Significant differences between the responses of WT and STAT4 − groups are indicated as follows: ***, P ≤ 0.0001; **, P ≤ 0.005.

    Article Snippet: For in vivo IFN-α treatment, LCMV-immune mice were i.v. injected with 5 × 105 U of IFN-α (PBL Interferon Source) at 90 min before harvest.

    Techniques: Infection, Mouse Assay, Flow Cytometry, Binding Assay, Ex Vivo, Staining

    A , Addition of IFN-α amplifies TLR7-stimulated proliferation and differentiation of CD27 + and CD27 − B cells. Isolated CD27 + and CD27 − CVID or control B cells were stimulated with 500 μmol/L loxoribine in the presence or

    Journal: The Journal of allergy and clinical immunology

    Article Title: Toll-like receptor 7 and 9 defects in common variable immunodeficiency

    doi: 10.1016/j.jaci.2009.05.019

    Figure Lengend Snippet: A , Addition of IFN-α amplifies TLR7-stimulated proliferation and differentiation of CD27 + and CD27 − B cells. Isolated CD27 + and CD27 − CVID or control B cells were stimulated with 500 μmol/L loxoribine in the presence or

    Article Snippet: Isolated CD19+ CD27+ and CD19+ CD27− B cell fractions were suspended in prewarmed PBS containing 0.5% BSA and labeled with 5-μmol/L carboxyfluorescein succinimidyl ester (Invitrogen, Carlsbad, Calif) for 5 minutes at room temperature, washed with 0.5% BSA/PBS, and cultured in complete medium with the optimum amount of loxoribine (500 μmol/L) , (InvivoGen, San Diego, Calif) in the presence or absence of an optimum concentration of IFN-α (1000 U/mL; Schering, Kenilworth, NJ).

    Techniques: Isolation

    A, Impaired production of IFN-α by TLR7-stimulated CVID PBMCs. CVID (n = 14–24) and control (n = 9–10) PBMCs were cultured with 10 μmol/L, 300 μmol/L, or 500 μmol/L loxoribine for 24 hours. B, Impaired production

    Journal: The Journal of allergy and clinical immunology

    Article Title: Toll-like receptor 7 and 9 defects in common variable immunodeficiency

    doi: 10.1016/j.jaci.2009.05.019

    Figure Lengend Snippet: A, Impaired production of IFN-α by TLR7-stimulated CVID PBMCs. CVID (n = 14–24) and control (n = 9–10) PBMCs were cultured with 10 μmol/L, 300 μmol/L, or 500 μmol/L loxoribine for 24 hours. B, Impaired production

    Article Snippet: Isolated CD19+ CD27+ and CD19+ CD27− B cell fractions were suspended in prewarmed PBS containing 0.5% BSA and labeled with 5-μmol/L carboxyfluorescein succinimidyl ester (Invitrogen, Carlsbad, Calif) for 5 minutes at room temperature, washed with 0.5% BSA/PBS, and cultured in complete medium with the optimum amount of loxoribine (500 μmol/L) , (InvivoGen, San Diego, Calif) in the presence or absence of an optimum concentration of IFN-α (1000 U/mL; Schering, Kenilworth, NJ).

    Techniques: Cell Culture

    a Increased STAT1 phosphorylation in patients’ monocytes after IFN-α and IFN-γ treatment. PBMC from patients (solid line) and healthy controls (dashed lines) were stimulated for 15 min with IFN-α (500 U/ml) or IFN-γ (100 ng/ml). Cells were fixed and permeabilized prior to staining with anti-CD14 and anti-phospho-STAT1 (pY701) antibodies. The gate was set on CD14+ monocytes. b Increased STAT1 phosphorylation in patients’ CD4+ T cells following IFN-α and IL-27 stimulation. PBMC were stimulated with either IFN-α (10,000 U/ml) or IL-27 (200 ng/ml) for 7.5, 15 and 30 min. Cells were then treated as described in ( a ) and phospho-STAT1 levels were evaluated gating on CD4+ T cells (Red – unstimulated patient cells; blue – stimulated healthy control cells; orange – stimulated patient cells)

    Journal: Journal of Clinical Immunology

    Article Title: The Extended Clinical Phenotype of 26 Patients with Chronic Mucocutaneous Candidiasis due to Gain-of-Function Mutations in STAT1

    doi: 10.1007/s10875-015-0214-9

    Figure Lengend Snippet: a Increased STAT1 phosphorylation in patients’ monocytes after IFN-α and IFN-γ treatment. PBMC from patients (solid line) and healthy controls (dashed lines) were stimulated for 15 min with IFN-α (500 U/ml) or IFN-γ (100 ng/ml). Cells were fixed and permeabilized prior to staining with anti-CD14 and anti-phospho-STAT1 (pY701) antibodies. The gate was set on CD14+ monocytes. b Increased STAT1 phosphorylation in patients’ CD4+ T cells following IFN-α and IL-27 stimulation. PBMC were stimulated with either IFN-α (10,000 U/ml) or IL-27 (200 ng/ml) for 7.5, 15 and 30 min. Cells were then treated as described in ( a ) and phospho-STAT1 levels were evaluated gating on CD4+ T cells (Red – unstimulated patient cells; blue – stimulated healthy control cells; orange – stimulated patient cells)

    Article Snippet: Intracellular monocyte staining: 1 × 106 PBMC were stimulated for 15 min at 37 °C with IFN-α (500 U/ml; Miltenyi Biotec) or IFN-γ (100 ng/ml; Miltenyi Biotec).

    Techniques: Staining

    pDC retain largely normal function in response to TLR7 stimulation during acute SIV infection. (A) Left: Contour plots demonstrating the gating strategy used to identify TNF-α + pDC in PBMC from SIV-naïve and SIV-infected monkeys following stimulation with 3M-007. Numbers represent the percentage of TNF-α + pDC within the indicated gates. Right: Percent of TNF-α + pDC in PBMC from SIV-naïve and SIV-infected monkeys in response to media or 3M-007. Bars represent the median and error bars the 95% confidence interval. (B) Left: Representative contour plots demonstrating the gating strategy used to identify TNF-α + , IFN-α + , or TNF-α + / IFN-α + pDC in lymph nodes following 3M-007 stimulation in the absence or presence of Brefeldin A (BFA). Numbers in parentheses represent the percentage of cells expressing the respective cytokines. Right: Percent of lymph node pDC from SIV-naïve and SIV-infected monkeys expressing TNF-α, IFN-α, or both in response to TLR7/8 agonist. Bars represent the median and error bars the 95% confidence interval. NS = not significant.

    Journal: PLoS Pathogens

    Article Title: Rapid Influx and Death of Plasmacytoid Dendritic Cells in Lymph Nodes Mediate Depletion in Acute Simian Immunodeficiency Virus Infection

    doi: 10.1371/journal.ppat.1000413

    Figure Lengend Snippet: pDC retain largely normal function in response to TLR7 stimulation during acute SIV infection. (A) Left: Contour plots demonstrating the gating strategy used to identify TNF-α + pDC in PBMC from SIV-naïve and SIV-infected monkeys following stimulation with 3M-007. Numbers represent the percentage of TNF-α + pDC within the indicated gates. Right: Percent of TNF-α + pDC in PBMC from SIV-naïve and SIV-infected monkeys in response to media or 3M-007. Bars represent the median and error bars the 95% confidence interval. (B) Left: Representative contour plots demonstrating the gating strategy used to identify TNF-α + , IFN-α + , or TNF-α + / IFN-α + pDC in lymph nodes following 3M-007 stimulation in the absence or presence of Brefeldin A (BFA). Numbers in parentheses represent the percentage of cells expressing the respective cytokines. Right: Percent of lymph node pDC from SIV-naïve and SIV-infected monkeys expressing TNF-α, IFN-α, or both in response to TLR7/8 agonist. Bars represent the median and error bars the 95% confidence interval. NS = not significant.

    Article Snippet: Cells were stained with surface-labeling antibodies and fixed and permeabilized as above prior to incubation with antibodies to TNF-α (MAb11) and/or IFN- α (MMHA-2, PBL Biomedical Laboratories).

    Techniques: Infection, Expressing

    A schematic of the mouse Batf2 gene, genotyping results, and the s.c. inoculated mouse tumor model. ( A ) A schematic of the mouse Batf2 gene, the targeting vector, and the targeted allele. ( B ) Genotyping PCR of Batf2 +/+ , Batf2 +/− , and Batf2 −/− mice. As indicated in A , primers shextra and wild together detect a fragment of the WT genomic locus, while primers shextra and PGKRC2 together detect a fragment of the mutant allele. ( C ) The relative expression levels of Batf2 mRNA in BMDMs from Batf2 −/− or WT littermates following stimulation by LPS (100 ng/mL) and IFN-γ (30 ng/mL) were quantified using qPCR ( Left ). The Batf2 mRNA levels in BMDMs from Batf2 −/− or WT littermates following stimulation with 10 3 U/mL of recombinant IFN-α were quantified using qPCR ( Right ). Error bars indicate ±SEM ( n = 2). ( D ) WT and Batf2 −/− mice were treated daily with imiquimod cream on shaved back skin for 6 d. The resulting erythema scores on days 4−6 are shown. Error bars indicate ±SEM ( n = 6). * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

    doi: 10.1073/pnas.1708598114

    Figure Lengend Snippet: A schematic of the mouse Batf2 gene, genotyping results, and the s.c. inoculated mouse tumor model. ( A ) A schematic of the mouse Batf2 gene, the targeting vector, and the targeted allele. ( B ) Genotyping PCR of Batf2 +/+ , Batf2 +/− , and Batf2 −/− mice. As indicated in A , primers shextra and wild together detect a fragment of the WT genomic locus, while primers shextra and PGKRC2 together detect a fragment of the mutant allele. ( C ) The relative expression levels of Batf2 mRNA in BMDMs from Batf2 −/− or WT littermates following stimulation by LPS (100 ng/mL) and IFN-γ (30 ng/mL) were quantified using qPCR ( Left ). The Batf2 mRNA levels in BMDMs from Batf2 −/− or WT littermates following stimulation with 10 3 U/mL of recombinant IFN-α were quantified using qPCR ( Right ). Error bars indicate ±SEM ( n = 2). ( D ) WT and Batf2 −/− mice were treated daily with imiquimod cream on shaved back skin for 6 d. The resulting erythema scores on days 4−6 are shown. Error bars indicate ±SEM ( n = 6). * P

    Article Snippet: BMDMs and BMDCs were stimulated with IFN-α (BioLegend), IFN-γ (R & D), or TLR ligands.

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Mouse Assay, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Recombinant

    IFN-α protein expression was significantly increased in lung after MAB microparticle challenge. (A) Shows a representative image of western blot bands, (B) shows significant difference in IFN-α protein expression in the lung of challenged mice (sample size 4 for each group).

    Journal: Frontiers in Immunology

    Article Title: Mycobacterium abscessus—Bronchial Epithelial Cells Cross-Talk Through Type I Interferon Signaling

    doi: 10.3389/fimmu.2019.02888

    Figure Lengend Snippet: IFN-α protein expression was significantly increased in lung after MAB microparticle challenge. (A) Shows a representative image of western blot bands, (B) shows significant difference in IFN-α protein expression in the lung of challenged mice (sample size 4 for each group).

    Article Snippet: Lungs were stained with antibodies against CD4 (rabbit, Abcam, catalogue# ab133616), CD8 (rabbit, Abcam, #ab12512), CD68 (rabbit, Abcam, #ab12512), PD-L1(rabbit, Proteintech, #17952-1-AP), and IFN-α (rabbit, Abcam, #ab193055) antibodies to identify infiltrating immune cells.

    Techniques: Expressing, Western Blot, Mouse Assay

    Development of non-caseating granuloma in the mouse lung after MAB microparticle challenge. (A,B) Black arrows show granulomas in the lung (HandE staining), (C) CD68, (D) CD4, (E) PD-L1 staining, (F) PD-1 staining, (G) IFN-α in challenged lung, (H) IFN-α in control. White arrow shows a group of positive cells for each staining. Magnification are x20 for all representative images. P -value shows percentage differences of lung stained cells between challenged mice and controls (sample size 3 controls and 4 challenged mice).

    Journal: Frontiers in Immunology

    Article Title: Mycobacterium abscessus—Bronchial Epithelial Cells Cross-Talk Through Type I Interferon Signaling

    doi: 10.3389/fimmu.2019.02888

    Figure Lengend Snippet: Development of non-caseating granuloma in the mouse lung after MAB microparticle challenge. (A,B) Black arrows show granulomas in the lung (HandE staining), (C) CD68, (D) CD4, (E) PD-L1 staining, (F) PD-1 staining, (G) IFN-α in challenged lung, (H) IFN-α in control. White arrow shows a group of positive cells for each staining. Magnification are x20 for all representative images. P -value shows percentage differences of lung stained cells between challenged mice and controls (sample size 3 controls and 4 challenged mice).

    Article Snippet: Lungs were stained with antibodies against CD4 (rabbit, Abcam, catalogue# ab133616), CD8 (rabbit, Abcam, #ab12512), CD68 (rabbit, Abcam, #ab12512), PD-L1(rabbit, Proteintech, #17952-1-AP), and IFN-α (rabbit, Abcam, #ab193055) antibodies to identify infiltrating immune cells.

    Techniques: Staining, Mouse Assay

    Immunostimulatory activity tests of Lys3-modivariants. Human PBMCs were stimulated with DOTAP encapsulated RNA species at indicated concentrations. Splint-ligated mv#1 and mv#2 containing half part of native tRNA Lys 3 and unmodified RNAs as well as native tRNA Lys 3 were tested on three individual donors in duplicate wells. IFN-α levels were measured in cell-free supernatants 20 h post-transfection. To account for donor variation results were normalized to the amount of IFN-α induced by 0.5 μg/ml bRNA. Each bar illustrates mean +SEM.

    Journal: Nucleic Acids Research

    Article Title: Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7

    doi: 10.1093/nar/gky644

    Figure Lengend Snippet: Immunostimulatory activity tests of Lys3-modivariants. Human PBMCs were stimulated with DOTAP encapsulated RNA species at indicated concentrations. Splint-ligated mv#1 and mv#2 containing half part of native tRNA Lys 3 and unmodified RNAs as well as native tRNA Lys 3 were tested on three individual donors in duplicate wells. IFN-α levels were measured in cell-free supernatants 20 h post-transfection. To account for donor variation results were normalized to the amount of IFN-α induced by 0.5 μg/ml bRNA. Each bar illustrates mean +SEM.

    Article Snippet: Cells were incubated in a humidified 5% CO2 atmosphere at 37°C for 20 h. Cell-free supernatants were analyzed by sandwich ELISA for secretion of IFN-α (Affymetrix eBioscience, Frankfurt, Germany) according to the manufacturer’s protocol.

    Techniques: Activity Assay, Transfection

    Native tRNA Lys3 and its immunostimulatory activity. ( A ) Human PBMCs were stimulated with DOTAP encapsulated bacterial RNA, an unmodified Lys3 tRNA and native tRNA Lys 3 (see Supplementary Figure S1A ) at three different concentrations (0.5, 0.25, 0.125 μg/ml). R848 (1 μg/ml) served as a positive control for TLR7 activation. IFN-α secretion was measured in cell-free supernatants after 20 h by ELISA. Shown bars represent the stimulation of 3–6 individual donors in duplicate wells +SEM. ( B ) To verify the antagonistic effect of native tRNA Lys 3 human PBMCs of three individual donors were co-transfected in duplicates with a constant amount of bacterial RNA (0.5 μg/ml) and different concentrations of the indicated RNAs (0.5, 0.25, 0.125, 0.0625, 0.03125 μg/ml). Previously described GmG and GG ORNs served as positive and negative control for a trans -inhibitory modification, respectively. To account for donor variation data was normalized to bacterial RNA. Dose-response model was calculated by R software as described in statistical analysis.

    Journal: Nucleic Acids Research

    Article Title: Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7

    doi: 10.1093/nar/gky644

    Figure Lengend Snippet: Native tRNA Lys3 and its immunostimulatory activity. ( A ) Human PBMCs were stimulated with DOTAP encapsulated bacterial RNA, an unmodified Lys3 tRNA and native tRNA Lys 3 (see Supplementary Figure S1A ) at three different concentrations (0.5, 0.25, 0.125 μg/ml). R848 (1 μg/ml) served as a positive control for TLR7 activation. IFN-α secretion was measured in cell-free supernatants after 20 h by ELISA. Shown bars represent the stimulation of 3–6 individual donors in duplicate wells +SEM. ( B ) To verify the antagonistic effect of native tRNA Lys 3 human PBMCs of three individual donors were co-transfected in duplicates with a constant amount of bacterial RNA (0.5 μg/ml) and different concentrations of the indicated RNAs (0.5, 0.25, 0.125, 0.0625, 0.03125 μg/ml). Previously described GmG and GG ORNs served as positive and negative control for a trans -inhibitory modification, respectively. To account for donor variation data was normalized to bacterial RNA. Dose-response model was calculated by R software as described in statistical analysis.

    Article Snippet: Cells were incubated in a humidified 5% CO2 atmosphere at 37°C for 20 h. Cell-free supernatants were analyzed by sandwich ELISA for secretion of IFN-α (Affymetrix eBioscience, Frankfurt, Germany) according to the manufacturer’s protocol.

    Techniques: Activity Assay, Positive Control, Activation Assay, Enzyme-linked Immunosorbent Assay, Transfection, Negative Control, Modification, Software

    Immunostimulation of single modified tRNAs. ( A ) Isolated PBMCs were stimulated with indicated tRNAs encapsulated with DOTAP at a concentration of 0.5, 0.25 and 0.125 μg/ml. Cell-free supernatants were collected after 20 h and IFN-α release was detected by ELISA. Linear regression was calculated by GraphPad Prism. ( B ) Transfection of single modified tRNAs incorporating indicated modifications at position 54 of tRNA sequence at a final concentration of 0.25 μg/ml. R848 and bRNA were used as a positive control for TLR7 stimulation. IFN-α secretion was compared to unmodified tRNA control. All stimulations were performed on three individual donors in duplicate wells. Shown are mean + SEM.

    Journal: Nucleic Acids Research

    Article Title: Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7

    doi: 10.1093/nar/gky644

    Figure Lengend Snippet: Immunostimulation of single modified tRNAs. ( A ) Isolated PBMCs were stimulated with indicated tRNAs encapsulated with DOTAP at a concentration of 0.5, 0.25 and 0.125 μg/ml. Cell-free supernatants were collected after 20 h and IFN-α release was detected by ELISA. Linear regression was calculated by GraphPad Prism. ( B ) Transfection of single modified tRNAs incorporating indicated modifications at position 54 of tRNA sequence at a final concentration of 0.25 μg/ml. R848 and bRNA were used as a positive control for TLR7 stimulation. IFN-α secretion was compared to unmodified tRNA control. All stimulations were performed on three individual donors in duplicate wells. Shown are mean + SEM.

    Article Snippet: Cells were incubated in a humidified 5% CO2 atmosphere at 37°C for 20 h. Cell-free supernatants were analyzed by sandwich ELISA for secretion of IFN-α (Affymetrix eBioscience, Frankfurt, Germany) according to the manufacturer’s protocol.

    Techniques: Modification, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Sequencing, Positive Control

    NV RNA replication is sensitive to type I and III IFN treatment. (A) Effect of type I and III IFN pretreatment on NV RNA replication. 293FT cells were pretreated with increasing doses of type I (100 and 1,000 U/ml IFN-α or IFN-β) or III (10 and 100 ng/ml IL-29) IFN for 24 h and then transfected with carrier RNA (−) or NV RNA (+) and incubated for 48 h. NV VP1 in cell lysate was detected by Western blotting. Actin served as an equal loading control. (B) Effect of posttransfection IFN treatment on NV RNA replication. 293FT cells were transfected with NV RNA and treated with 1,000 U/ml IFN-β at 12 h post-RNA transfection. Cells were lysed at 48 h post-RNA transfection, and NV VP1 in cell lysate was detected by IP and Western blotting. (C) Immunofluorescence staining of NV VP1 in NV RNA-transfected 293FT cells either untreated (no IFN) or pretreated with 1,000 U/ml IFN-β. Cells were fixed at 48 h post-RNA transfection and stained with guinea pig antibody to NV VP1. Scale bars, 200 μm.

    Journal: Journal of Virology

    Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

    doi: 10.1128/JVI.01425-16

    Figure Lengend Snippet: NV RNA replication is sensitive to type I and III IFN treatment. (A) Effect of type I and III IFN pretreatment on NV RNA replication. 293FT cells were pretreated with increasing doses of type I (100 and 1,000 U/ml IFN-α or IFN-β) or III (10 and 100 ng/ml IL-29) IFN for 24 h and then transfected with carrier RNA (−) or NV RNA (+) and incubated for 48 h. NV VP1 in cell lysate was detected by Western blotting. Actin served as an equal loading control. (B) Effect of posttransfection IFN treatment on NV RNA replication. 293FT cells were transfected with NV RNA and treated with 1,000 U/ml IFN-β at 12 h post-RNA transfection. Cells were lysed at 48 h post-RNA transfection, and NV VP1 in cell lysate was detected by IP and Western blotting. (C) Immunofluorescence staining of NV VP1 in NV RNA-transfected 293FT cells either untreated (no IFN) or pretreated with 1,000 U/ml IFN-β. Cells were fixed at 48 h post-RNA transfection and stained with guinea pig antibody to NV VP1. Scale bars, 200 μm.

    Article Snippet: IFN-α (PHP108Z; AbD Serotec) and IFN-β (407318; CalBiochem) were used at 1,000 U/ml unless otherwise indicated.

    Techniques: Transfection, Incubation, Western Blot, Immunofluorescence, Staining

    NV RNA replication does not induce an IFN response. (A) 293FT-ISRE-Luc reporter cells have strong IFN responses to various stimuli and detect both IFN induction and signaling. Cells were pretreated with B18R protein (125 ng/ml) or carrier protein BSA (control) for 1 h and stimulated with the indicated reagents for 18 h. Doses of stimuli were as follows: SeV, 40 HA/ml; poly(I·C), 100 μg/ml; IFN-α and IFN-β, 1,000 U/ml; IL-29, 100 ng/ml. Luciferase activity was normalized to that of BSA-pretreated and unstimulated (mock) cells and is presented as fold induction. *, P

    Journal: Journal of Virology

    Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

    doi: 10.1128/JVI.01425-16

    Figure Lengend Snippet: NV RNA replication does not induce an IFN response. (A) 293FT-ISRE-Luc reporter cells have strong IFN responses to various stimuli and detect both IFN induction and signaling. Cells were pretreated with B18R protein (125 ng/ml) or carrier protein BSA (control) for 1 h and stimulated with the indicated reagents for 18 h. Doses of stimuli were as follows: SeV, 40 HA/ml; poly(I·C), 100 μg/ml; IFN-α and IFN-β, 1,000 U/ml; IL-29, 100 ng/ml. Luciferase activity was normalized to that of BSA-pretreated and unstimulated (mock) cells and is presented as fold induction. *, P

    Article Snippet: IFN-α (PHP108Z; AbD Serotec) and IFN-β (407318; CalBiochem) were used at 1,000 U/ml unless otherwise indicated.

    Techniques: Luciferase, Activity Assay

    NV RNA replication is not enhanced by neutralization of type I IFNs. (A) Western blotting of NV VP1 in 293FT cells pretreated with type I IFN neutralizing antibodies for 6 h and transfected with carrier RNA or NV RNA for 48 h. See Materials and Methods for the dilutions of the antibodies. Sh, sheep; Rb, rabbit; Ab, antibody. (B and C) Western blotting of NV VP1 and VPg in 293FT cells pretreated with BSA (−) or with 125 ng/ml B18R (+) for 1 h and transfected with carrier RNA or NV RNA for 48 h. Actin served as an equal loading control . (D) 293FT-ISRE-Luc reporter assay of type I IFN neutralizing antibodies. Spent medium (containing IFN neutralizing antibody) from the experiment shown in panel A was collected before lysis of cells and added to 293FT-ISRE-Luc reporter cells for 1 h, followed by stimulation with 500 U/ml IFN-α or IFN-β for 18 h. Luciferase activity (presented as fold induction) was normalized to that of the reporter cells that received medium from untreated (no antibody and no IFN) carrier RNA-transfected 293FT cells. * and ^, P

    Journal: Journal of Virology

    Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

    doi: 10.1128/JVI.01425-16

    Figure Lengend Snippet: NV RNA replication is not enhanced by neutralization of type I IFNs. (A) Western blotting of NV VP1 in 293FT cells pretreated with type I IFN neutralizing antibodies for 6 h and transfected with carrier RNA or NV RNA for 48 h. See Materials and Methods for the dilutions of the antibodies. Sh, sheep; Rb, rabbit; Ab, antibody. (B and C) Western blotting of NV VP1 and VPg in 293FT cells pretreated with BSA (−) or with 125 ng/ml B18R (+) for 1 h and transfected with carrier RNA or NV RNA for 48 h. Actin served as an equal loading control . (D) 293FT-ISRE-Luc reporter assay of type I IFN neutralizing antibodies. Spent medium (containing IFN neutralizing antibody) from the experiment shown in panel A was collected before lysis of cells and added to 293FT-ISRE-Luc reporter cells for 1 h, followed by stimulation with 500 U/ml IFN-α or IFN-β for 18 h. Luciferase activity (presented as fold induction) was normalized to that of the reporter cells that received medium from untreated (no antibody and no IFN) carrier RNA-transfected 293FT cells. * and ^, P

    Article Snippet: IFN-α (PHP108Z; AbD Serotec) and IFN-β (407318; CalBiochem) were used at 1,000 U/ml unless otherwise indicated.

    Techniques: Neutralization, Western Blot, Transfection, Reporter Assay, Lysis, Luciferase, Activity Assay

    Capture of SUMOylated PML and p53 using SUBE-l. NB4 cells (A) or MCF7 (B) were pre-treated with MG-132 before 1 h of ATO treatment. (C) SUMO and PML can be co-localized in the nuclear bodies (NB) of NB4 cells under the same conditions than (A or B). SUMOylated p53 can be captured by SUBE-l (D). HEK-293 were untreated or treated with IFN-α or infected with VSV. Input, flow-through (FT) and bound fractions were analysed with anti-SUMO-2/-3, PML or p53 antibodies. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as loading control.

    Journal: Scientific Reports

    Article Title: Analysis of SUMOylated proteins using SUMO-traps

    doi: 10.1038/srep01690

    Figure Lengend Snippet: Capture of SUMOylated PML and p53 using SUBE-l. NB4 cells (A) or MCF7 (B) were pre-treated with MG-132 before 1 h of ATO treatment. (C) SUMO and PML can be co-localized in the nuclear bodies (NB) of NB4 cells under the same conditions than (A or B). SUMOylated p53 can be captured by SUBE-l (D). HEK-293 were untreated or treated with IFN-α or infected with VSV. Input, flow-through (FT) and bound fractions were analysed with anti-SUMO-2/-3, PML or p53 antibodies. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as loading control.

    Article Snippet: HEK-293 were transfected with His6-SUMO-2 protein and were treated with 500 U/ml of IFN-α (GenScript) for 24 h, infected with Indiana strain VSV at an MOI of 1 PFU/ml for 4 h, or left untreated.

    Techniques: Infection, Flow Cytometry

    2'-O-methylated RNA inhibits IFN-α, TNF and IL-12p40 production with similar efficiency. a-c PBMCs were cotransfected overnight with 1 µg/ml bRNA purified from S. aureus and different ratios of 2'-O-methylated inhibitory RNA (inh. RNA). Levels of IFN-α ( a ), TNF ( b ) and IL-12p40 ( c ) were analysed in cell-free supernatants by ELISA. Data were normalized to cytokine production induced by bRNA alone to account for donor variation. Curve fit and IC 50 ( d ) were calculated with R software using the 4-parameter Weibull function. Each data point represents the average value of 2–6 independent experiments with different donors.

    Journal: Journal of Innate Immunity

    Article Title: 2'-O-Methylation within Bacterial RNA Acts as Suppressor of TLR7/TLR8 Activation in Human Innate Immune Cells

    doi: 10.1159/000375460

    Figure Lengend Snippet: 2'-O-methylated RNA inhibits IFN-α, TNF and IL-12p40 production with similar efficiency. a-c PBMCs were cotransfected overnight with 1 µg/ml bRNA purified from S. aureus and different ratios of 2'-O-methylated inhibitory RNA (inh. RNA). Levels of IFN-α ( a ), TNF ( b ) and IL-12p40 ( c ) were analysed in cell-free supernatants by ELISA. Data were normalized to cytokine production induced by bRNA alone to account for donor variation. Curve fit and IC 50 ( d ) were calculated with R software using the 4-parameter Weibull function. Each data point represents the average value of 2–6 independent experiments with different donors.

    Article Snippet: For cytokine measurement, the levels of TNF, IL-6, IL-12p40 (BD, Heidelberg, Germany) and IFN-α (Bender Med Systems, Vienna, Austria) were determined in cell-free supernatants by ELISA.

    Techniques: Methylation, Purification, Enzyme-linked Immunosorbent Assay, Software

    Comparison of protein microarray performance with an established bead-based assay. A , Corresponding MFI values from protein microarrays and a bead-based assay were plotted for the targets GM-CSF and IFN-α, and Pearson correlation coefficients

    Journal: The Journal of allergy and clinical immunology

    Article Title: Protein microarrays identify disease-specific anti-cytokine autoantibody profiles in the landscape of immunodeficiency

    doi: 10.1016/j.jaci.2015.07.032

    Figure Lengend Snippet: Comparison of protein microarray performance with an established bead-based assay. A , Corresponding MFI values from protein microarrays and a bead-based assay were plotted for the targets GM-CSF and IFN-α, and Pearson correlation coefficients

    Article Snippet: IFN-α (2000 IU per well, INTRONA; Merck, Kenilworth, NJ) or LPS (1 µg/mL; BioLabs, ■■■) alone or both stimuli were added.

    Techniques: Microarray, Bead-based Assay

    TRAIL induction is driven primarily by cytokines. (A) PBMC were infected with influenza. After infection, decreasing numbers of cells (10 6 – 1.25 × 10 5 cells) were aliquoted into 2 ml of media. After 24 h culture, IFN-α levels

    Journal:

    Article Title: Influenza-induced expression of functional TNF-related apoptosis-inducing ligand (TRAIL) on human PBMC

    doi: 10.1016/j.humimm.2008.07.012

    Figure Lengend Snippet: TRAIL induction is driven primarily by cytokines. (A) PBMC were infected with influenza. After infection, decreasing numbers of cells (10 6 – 1.25 × 10 5 cells) were aliquoted into 2 ml of media. After 24 h culture, IFN-α levels

    Article Snippet: As a positive control for inducing TRAIL expression, uninfected PBMC were treated with 500ng/ml IFN-α (Cell Signaling Technologies, Danvers, MA) for 24 h.

    Techniques: Infection

    Influenza stimulates IFN-α and –γ production from PBMC. (A) PBMC were infected with viable or UV-inactivated influenza (UV-flu) or stimulated with the TLR agonists poly I:C, ssRNA, or CpG ODN. After 24 h, IFN-α and –γ

    Journal:

    Article Title: Influenza-induced expression of functional TNF-related apoptosis-inducing ligand (TRAIL) on human PBMC

    doi: 10.1016/j.humimm.2008.07.012

    Figure Lengend Snippet: Influenza stimulates IFN-α and –γ production from PBMC. (A) PBMC were infected with viable or UV-inactivated influenza (UV-flu) or stimulated with the TLR agonists poly I:C, ssRNA, or CpG ODN. After 24 h, IFN-α and –γ

    Article Snippet: As a positive control for inducing TRAIL expression, uninfected PBMC were treated with 500ng/ml IFN-α (Cell Signaling Technologies, Danvers, MA) for 24 h.

    Techniques: Infection

    Expression of IFN-α and TNF-α in A498 xenografts after rIL-22 treatment. Western blot assay was performed to detect the expression of IFN-α and TNF-α in the A498 cell xenografts. Neither the expression of IFN-α nor TNF-α were increased significantly in A498 cell xenografts treated with rIL-22 (P > 0.05 compared with control). N = 2.

    Journal: PLoS ONE

    Article Title: Interleukin-22 Suppresses the Growth of A498 Renal Cell Carcinoma Cells via Regulation of STAT1 Pathway

    doi: 10.1371/journal.pone.0020382

    Figure Lengend Snippet: Expression of IFN-α and TNF-α in A498 xenografts after rIL-22 treatment. Western blot assay was performed to detect the expression of IFN-α and TNF-α in the A498 cell xenografts. Neither the expression of IFN-α nor TNF-α were increased significantly in A498 cell xenografts treated with rIL-22 (P > 0.05 compared with control). N = 2.

    Article Snippet: The membrane was immersed in blocking buffer (5% non-fat dry milk/1% Tween 20 in 20 mM TBS, pH 7.5) for 1.5 h at room temperature and incubated with the appropriate primary antibodies, mouse monoclonal anti-TNF-α (1∶ 50) and IFN-α (1∶100) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in blocking buffer overnight at 4°C, followed by incubation with complementary secondary antibody.

    Techniques: Expressing, Western Blot

    Activation of mouse-derived dendritic cells (DCs) by LNP-CpGs in vitro . Mouse-derived DCs were treated with CpG ODN or with each LNP-CpG for 24 h in vitro . The CpG ODN content was the same between the cells treated with CpG ODN alone and those treated with LNP-CpGs. (A) Cytokine production. Levels of IL-12 p40 and IFN-α in the supernatants were measured by ELISA. (B) Expression of co-stimulatory molecules. Expression of CD80 and CD86 on DCs was measured by flow cytometry; percentages of positive DCs are shown. (A,B) n = 5 per group. Data are means ± SD. † P

    Journal: Frontiers in Immunology

    Article Title: Lipid Nanoparticles Potentiate CpG-Oligodeoxynucleotide-Based Vaccine for Influenza Virus

    doi: 10.3389/fimmu.2019.03018

    Figure Lengend Snippet: Activation of mouse-derived dendritic cells (DCs) by LNP-CpGs in vitro . Mouse-derived DCs were treated with CpG ODN or with each LNP-CpG for 24 h in vitro . The CpG ODN content was the same between the cells treated with CpG ODN alone and those treated with LNP-CpGs. (A) Cytokine production. Levels of IL-12 p40 and IFN-α in the supernatants were measured by ELISA. (B) Expression of co-stimulatory molecules. Expression of CD80 and CD86 on DCs was measured by flow cytometry; percentages of positive DCs are shown. (A,B) n = 5 per group. Data are means ± SD. † P

    Article Snippet: These cells were stimulated with CpG ODN or with each LNP-CpG for 24 h. Supernatants were subjected to ELISA to determine the levels of IFN-α (InvivoGen) and interleukin (IL)-12 p40 (BioLegend, San Diego, CA, USA), in accordance with the manufacturers' instructions.

    Techniques: Activation Assay, Derivative Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

    IFN-α-induced Trex1 expression is IFNAR1-, STAT1-, and STAT2-dependent.

    Journal: Journal of Leukocyte Biology

    Article Title: TLR ligands up-regulate Trex1 expression in murine conventional dendritic cells through type I Interferon and NF-κB-dependent signaling pathways

    doi: 10.1189/jlb.2A0713-393RR

    Figure Lengend Snippet: IFN-α-induced Trex1 expression is IFNAR1-, STAT1-, and STAT2-dependent.

    Article Snippet: Resting cDC cultures were stimulated on Day 6 or 7 of culture with the following stimuli: 1500 U/ml IFN-α (HyCult Biotechnology, PB Uden, the Netherlands), 100 ng/ml LPS (Sigma-Aldrich, St. Louis, MO, USA), 10 μg/ml CpG-B 1826 (IDT Biotechnologies, Coralville, IA, USA), 1 μg/ml R848 (Invivogen, San Diego, CA, USA), and 200 ng/ml PolyI:C (Alexis Biochemicals, San Diego, CA, USA).

    Techniques: Expressing

    Opn-dependent in vivo response to HSV-1 infection. ( a ) IFN-α concentrations from Opn wild-type (WT) and Opn-deficient (KO) pDC culture supernatants after incubation in vitro with ultraviolet irradiation–treated (UV-irradiated) HSV-1 for

    Journal:

    Article Title: Osteopontin expression is essential for interferon-? production by plasmacytoid dendritic cells

    doi: 10.1038/ni1327

    Figure Lengend Snippet: Opn-dependent in vivo response to HSV-1 infection. ( a ) IFN-α concentrations from Opn wild-type (WT) and Opn-deficient (KO) pDC culture supernatants after incubation in vitro with ultraviolet irradiation–treated (UV-irradiated) HSV-1 for

    Article Snippet: Primers were as follows: Opn forward, 5′-GCCTGTTTGGCATTGCCTCCTC-3′, and reverse, 5′-CACAGCATTCTGTGGCGCAAGG-3′; β-actin forward, 5′-TGTTACCAACTGGGACGACA-3′, and reverse, 5′-CTGGGTCATCTTTTCACGGT-3′; primer sequences for detecting subsets of IFN-α were obtained from Clontech.

    Techniques: In Vivo, Infection, Incubation, In Vitro, Irradiation

    IFN-α-dependent cross-presentation. ( a , b ) Purified bone marrow–derived Opn-deficient (Opn KO) and Opn wild-type (Opn WT) pDCs were treated with or without CpG-B (0.2 μg/ml) and/or recombinant IFN-α (rIFN-α; 1 ×

    Journal:

    Article Title: Osteopontin expression is essential for interferon-? production by plasmacytoid dendritic cells

    doi: 10.1038/ni1327

    Figure Lengend Snippet: IFN-α-dependent cross-presentation. ( a , b ) Purified bone marrow–derived Opn-deficient (Opn KO) and Opn wild-type (Opn WT) pDCs were treated with or without CpG-B (0.2 μg/ml) and/or recombinant IFN-α (rIFN-α; 1 ×

    Article Snippet: Primers were as follows: Opn forward, 5′-GCCTGTTTGGCATTGCCTCCTC-3′, and reverse, 5′-CACAGCATTCTGTGGCGCAAGG-3′; β-actin forward, 5′-TGTTACCAACTGGGACGACA-3′, and reverse, 5′-CTGGGTCATCTTTTCACGGT-3′; primer sequences for detecting subsets of IFN-α were obtained from Clontech.

    Techniques: Purification, Derivative Assay, Recombinant

    Cytokine production by pDCs. ( a ) IFN-α (left) and IL-6 (right) in 24-hour pDC culture supernatants of T-bet wild-type (T-bet WT) versus T-bet-deficient (T-bet KO) cells stimulated with various concentrations of CpG-B (ODN-1668). The pDCs were

    Journal:

    Article Title: Osteopontin expression is essential for interferon-? production by plasmacytoid dendritic cells

    doi: 10.1038/ni1327

    Figure Lengend Snippet: Cytokine production by pDCs. ( a ) IFN-α (left) and IL-6 (right) in 24-hour pDC culture supernatants of T-bet wild-type (T-bet WT) versus T-bet-deficient (T-bet KO) cells stimulated with various concentrations of CpG-B (ODN-1668). The pDCs were

    Article Snippet: Primers were as follows: Opn forward, 5′-GCCTGTTTGGCATTGCCTCCTC-3′, and reverse, 5′-CACAGCATTCTGTGGCGCAAGG-3′; β-actin forward, 5′-TGTTACCAACTGGGACGACA-3′, and reverse, 5′-CTGGGTCATCTTTTCACGGT-3′; primer sequences for detecting subsets of IFN-α were obtained from Clontech.

    Techniques:

    Intracellular Opn is required for IFN-α production. ( a – c ) Lentivirus infection of pDCs stimulated with CpG-B (0.2 μg/ml). ( a ) Extracellular Opn protein measured by ELISA in 6-hour culture supernatants of sorted splenic Opn-deficient

    Journal:

    Article Title: Osteopontin expression is essential for interferon-? production by plasmacytoid dendritic cells

    doi: 10.1038/ni1327

    Figure Lengend Snippet: Intracellular Opn is required for IFN-α production. ( a – c ) Lentivirus infection of pDCs stimulated with CpG-B (0.2 μg/ml). ( a ) Extracellular Opn protein measured by ELISA in 6-hour culture supernatants of sorted splenic Opn-deficient

    Article Snippet: Primers were as follows: Opn forward, 5′-GCCTGTTTGGCATTGCCTCCTC-3′, and reverse, 5′-CACAGCATTCTGTGGCGCAAGG-3′; β-actin forward, 5′-TGTTACCAACTGGGACGACA-3′, and reverse, 5′-CTGGGTCATCTTTTCACGGT-3′; primer sequences for detecting subsets of IFN-α were obtained from Clontech.

    Techniques: Infection, Enzyme-linked Immunosorbent Assay

    Blockade of IFN-α significantly reduces islet immunopathology in Rip -LCMV mice infected with virus. Rip- LCMV mice were treated with IgG isotype control antibody (1 mg; Clone PIP), antibody to IFNAR (1 mg; Clone: MAR1-5A3), antibody to IFN-α

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Progression of type 1 diabetes from the prediabetic stage is controlled by interferon-α signaling

    doi: 10.1073/pnas.1700878114

    Figure Lengend Snippet: Blockade of IFN-α significantly reduces islet immunopathology in Rip -LCMV mice infected with virus. Rip- LCMV mice were treated with IgG isotype control antibody (1 mg; Clone PIP), antibody to IFNAR (1 mg; Clone: MAR1-5A3), antibody to IFN-α

    Article Snippet: Rip- LCMV mice were treated with either IgG isotype control antibody (1 mg; Clone PIP; Leinco Technologies), antibody to IFNAR (1 mg; Clone: MAR1-5A3; Leinco Technologies), antibody to IFN-α (1 mg; Clone: TIF-3C5; Leinco Technologies) or antibody to IFN-β (0.25 mg; Clone: HDβ-4A7; Leinco Technologies) 1 d before infection with LCMV and at 5 d p.i.

    Techniques: Mouse Assay, Infection

    Blockade of IFN-α signaling prevents the migration of anti-self GP-specific T cells into the islets but does not limit their expansion or effector activity in the spleen. Twenty thousand P14 CD8 + T cells were adoptively transferred into Rip -LCMV

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Progression of type 1 diabetes from the prediabetic stage is controlled by interferon-α signaling

    doi: 10.1073/pnas.1700878114

    Figure Lengend Snippet: Blockade of IFN-α signaling prevents the migration of anti-self GP-specific T cells into the islets but does not limit their expansion or effector activity in the spleen. Twenty thousand P14 CD8 + T cells were adoptively transferred into Rip -LCMV

    Article Snippet: Rip- LCMV mice were treated with either IgG isotype control antibody (1 mg; Clone PIP; Leinco Technologies), antibody to IFNAR (1 mg; Clone: MAR1-5A3; Leinco Technologies), antibody to IFN-α (1 mg; Clone: TIF-3C5; Leinco Technologies) or antibody to IFN-β (0.25 mg; Clone: HDβ-4A7; Leinco Technologies) 1 d before infection with LCMV and at 5 d p.i.

    Techniques: Migration, Activity Assay

    Neutralization of IFN-α by antibody blockade did not induce transcriptional changes within the pancreatic islets of Rip -LCMV mice at day 3 p.i. Rip -LCMV mice were treated with anti–IFN-α or IgG isotype control and killed at day

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Progression of type 1 diabetes from the prediabetic stage is controlled by interferon-α signaling

    doi: 10.1073/pnas.1700878114

    Figure Lengend Snippet: Neutralization of IFN-α by antibody blockade did not induce transcriptional changes within the pancreatic islets of Rip -LCMV mice at day 3 p.i. Rip -LCMV mice were treated with anti–IFN-α or IgG isotype control and killed at day

    Article Snippet: Rip- LCMV mice were treated with either IgG isotype control antibody (1 mg; Clone PIP; Leinco Technologies), antibody to IFNAR (1 mg; Clone: MAR1-5A3; Leinco Technologies), antibody to IFN-α (1 mg; Clone: TIF-3C5; Leinco Technologies) or antibody to IFN-β (0.25 mg; Clone: HDβ-4A7; Leinco Technologies) 1 d before infection with LCMV and at 5 d p.i.

    Techniques: Neutralization, Mouse Assay

    IFN-α signaling is required for development of T1D. Rip -LCMV mice were infected with 2 × 10 5 pfu LCMV i.p. and treated with either IgG isotype control antibody or antibody to IFNAR, to IFN-α or IFN-β at day −1 and

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Progression of type 1 diabetes from the prediabetic stage is controlled by interferon-α signaling

    doi: 10.1073/pnas.1700878114

    Figure Lengend Snippet: IFN-α signaling is required for development of T1D. Rip -LCMV mice were infected with 2 × 10 5 pfu LCMV i.p. and treated with either IgG isotype control antibody or antibody to IFNAR, to IFN-α or IFN-β at day −1 and

    Article Snippet: Rip- LCMV mice were treated with either IgG isotype control antibody (1 mg; Clone PIP; Leinco Technologies), antibody to IFNAR (1 mg; Clone: MAR1-5A3; Leinco Technologies), antibody to IFN-α (1 mg; Clone: TIF-3C5; Leinco Technologies) or antibody to IFN-β (0.25 mg; Clone: HDβ-4A7; Leinco Technologies) 1 d before infection with LCMV and at 5 d p.i.

    Techniques: Mouse Assay, Infection

    Blockade of IFN-α increases the expression of CD8 + T-cell effector genes within P14 T cells. P14 T cells were sorted by FACS from the spleens at day 7 p.i. and subject to RNA-seq analysis. ( A ) We identified 41 differentially expressed genes (FDR

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Progression of type 1 diabetes from the prediabetic stage is controlled by interferon-α signaling

    doi: 10.1073/pnas.1700878114

    Figure Lengend Snippet: Blockade of IFN-α increases the expression of CD8 + T-cell effector genes within P14 T cells. P14 T cells were sorted by FACS from the spleens at day 7 p.i. and subject to RNA-seq analysis. ( A ) We identified 41 differentially expressed genes (FDR

    Article Snippet: Rip- LCMV mice were treated with either IgG isotype control antibody (1 mg; Clone PIP; Leinco Technologies), antibody to IFNAR (1 mg; Clone: MAR1-5A3; Leinco Technologies), antibody to IFN-α (1 mg; Clone: TIF-3C5; Leinco Technologies) or antibody to IFN-β (0.25 mg; Clone: HDβ-4A7; Leinco Technologies) 1 d before infection with LCMV and at 5 d p.i.

    Techniques: Expressing, FACS, RNA Sequencing Assay

    Effect of IAV H3N2 NS1 protein mutations on innate immune responses induced by SeV infection. Human 293T cells were transiently transfected using the calcium phosphate method with the indicated pCAGGS NS1-expressing plasmids, together with plasmids expressing Fluc under the control of an IFN-β (A) or ISRE promoter (B). At 24 hpt, cells were mock infected (M) or infected with SeV (Cantell strain) (+SeV) to induce activation of the promoters, and 16 hpi cell lysates were prepared for reporter gene expression. (A and B) Reporter Firefly expression was measured by luminescence. Data represented show the means and standard deviations of the results determined for triplicate wells. Experiments were repeated 3 times in triplicate wells with similar results. P values determined using Student's t test are indicated. (C) At 16 h after SeV infection, TCS were collected and, after UV inactivation, were used to treat fresh A549 cells. Alternatively, A549 cells were treated with 2,500 U/ml of universal IFN-α as a control (picture on the right). After 24 h of incubation, cells were infected (MOI, 0.001) with the IFN-sensitive VSV-GFP. At 16 hpi, VSV-GFP-infected cells were observed under a fluorescence microscope. (D) At 16 h after SeV infection, cellular extracts were obtained and the expression of ISG15 and actin were evaluated by Western blotting, using antibodies specific to ISG15 and to actin (as a loading control). Western blots were quantified by densitometry using the software ImageJ (v1.46), and the amounts of ISG15 protein were normalized to the amounts of actin protein (numbers below the ISG15 blot; ND, not detected). Molecular mass markers (in kilodaltons) are indicated on the right. The squares represent the NS1 variants found in the subjects ( Table 4 ), showing the numbers of patients for each NS1 variant below the squares.

    Journal: Journal of Virology

    Article Title: NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses

    doi: 10.1128/JVI.01039-16

    Figure Lengend Snippet: Effect of IAV H3N2 NS1 protein mutations on innate immune responses induced by SeV infection. Human 293T cells were transiently transfected using the calcium phosphate method with the indicated pCAGGS NS1-expressing plasmids, together with plasmids expressing Fluc under the control of an IFN-β (A) or ISRE promoter (B). At 24 hpt, cells were mock infected (M) or infected with SeV (Cantell strain) (+SeV) to induce activation of the promoters, and 16 hpi cell lysates were prepared for reporter gene expression. (A and B) Reporter Firefly expression was measured by luminescence. Data represented show the means and standard deviations of the results determined for triplicate wells. Experiments were repeated 3 times in triplicate wells with similar results. P values determined using Student's t test are indicated. (C) At 16 h after SeV infection, TCS were collected and, after UV inactivation, were used to treat fresh A549 cells. Alternatively, A549 cells were treated with 2,500 U/ml of universal IFN-α as a control (picture on the right). After 24 h of incubation, cells were infected (MOI, 0.001) with the IFN-sensitive VSV-GFP. At 16 hpi, VSV-GFP-infected cells were observed under a fluorescence microscope. (D) At 16 h after SeV infection, cellular extracts were obtained and the expression of ISG15 and actin were evaluated by Western blotting, using antibodies specific to ISG15 and to actin (as a loading control). Western blots were quantified by densitometry using the software ImageJ (v1.46), and the amounts of ISG15 protein were normalized to the amounts of actin protein (numbers below the ISG15 blot; ND, not detected). Molecular mass markers (in kilodaltons) are indicated on the right. The squares represent the NS1 variants found in the subjects ( Table 4 ), showing the numbers of patients for each NS1 variant below the squares.

    Article Snippet: In the experiments to measure IFN sensitivity, the medium also contained 2,000 U/ml of universal IFN-α (Axxora).

    Techniques: Infection, Transfection, Expressing, Activation Assay, Incubation, Fluorescence, Microscopy, Western Blot, Software, Variant Assay

    Induction of IFN responses on PBMCs from infected subjects. PBMCs from subject 85 (infected with the virus encoding T64-NS1; in gray) and from subjects 64, 68, 87, 21, 23, and 44 (infected with viruses encoding I64-NS1; in black) were either treated with 2,000 U/ml of IFN-α (A) or infected (MOI, 1) with virus r85 (B). The levels of mRNA expression of cellular IFIT2 (A), IFN-λ1 (B), or gene M mRNA in the infected PBMCs (C) were analyzed by qRT-PCR. Bars represent standard deviations of the means from triplicates. P values determined using Student's t test are indicated. The experiments were repeated twice independently, using technical triplicates, with similar results. r.u., relative units.

    Journal: Journal of Virology

    Article Title: NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses

    doi: 10.1128/JVI.01039-16

    Figure Lengend Snippet: Induction of IFN responses on PBMCs from infected subjects. PBMCs from subject 85 (infected with the virus encoding T64-NS1; in gray) and from subjects 64, 68, 87, 21, 23, and 44 (infected with viruses encoding I64-NS1; in black) were either treated with 2,000 U/ml of IFN-α (A) or infected (MOI, 1) with virus r85 (B). The levels of mRNA expression of cellular IFIT2 (A), IFN-λ1 (B), or gene M mRNA in the infected PBMCs (C) were analyzed by qRT-PCR. Bars represent standard deviations of the means from triplicates. P values determined using Student's t test are indicated. The experiments were repeated twice independently, using technical triplicates, with similar results. r.u., relative units.

    Article Snippet: In the experiments to measure IFN sensitivity, the medium also contained 2,000 U/ml of universal IFN-α (Axxora).

    Techniques: Infection, Expressing, Quantitative RT-PCR

    Recombinant virus growth kinetics in vitro . (A) Scheme of the pDZ plasmid encoding the NS split segment ( 27 ) used to rescue the viruses r65 and r85 (top lane) as well as the NS segment encoding the NS1 and NEP proteins (second lane) being processed by the porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site (third lane). Viral 3′ and 5′ noncoding regions are indicated with black boxes at the end of the viral segment. Viral products from the NS (NS1 and NEP) segment are indicated with white boxes. The region before the splicing donor in the viral segments is indicated with light gray boxes. The sequence of the PTV-1 2A autoproteolytic cleavage site (2A) is indicated with dark gray boxes. The sequences encoding the polymerase (Pol) I and II promoters, the Pol I promoter terminator (T), and the poly(A) tail (pA) are indicated. The position of restriction sites EcoRI and BsmBI, used to clone the NS1 sequences from patients 65 and 85, is shown. (B) Canine MDCK (left) and human A549 (right) cells were infected in duplicate with recombinant PR8 viruses expressing either 65 and 85 H3N2 NS1 proteins or the PR8 NS1 protein (wt-PR8-NSs) at an MOI of 0.001. Virus titers in infected cell supernatants were determined at different times postinfection by immunofocus assay. (C) MDCK cells were infected (MOI, 0.001) with viruses r65 and r85, and just after the infection, cells were left untreated or were treated with 2,000 U of IFN-α/well. Supernatants were collected at 24 and 48 hpi, and virus titers were determined by immunofocus assay. (Left) The percentage of growth in MDCK cells treated with IFN-α (+) was normalized to the growth in the nontreated cells (−) at 24 and 48 hpi (considered 100% growth). (Right) Virus titers (in FFU/ml) in nontreated (−) and IFN-treated (+) cells are shown. The experiments were repeated 3 times in duplicate wells. The dotted line in panel B indicates the limit of detection.

    Journal: Journal of Virology

    Article Title: NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses

    doi: 10.1128/JVI.01039-16

    Figure Lengend Snippet: Recombinant virus growth kinetics in vitro . (A) Scheme of the pDZ plasmid encoding the NS split segment ( 27 ) used to rescue the viruses r65 and r85 (top lane) as well as the NS segment encoding the NS1 and NEP proteins (second lane) being processed by the porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site (third lane). Viral 3′ and 5′ noncoding regions are indicated with black boxes at the end of the viral segment. Viral products from the NS (NS1 and NEP) segment are indicated with white boxes. The region before the splicing donor in the viral segments is indicated with light gray boxes. The sequence of the PTV-1 2A autoproteolytic cleavage site (2A) is indicated with dark gray boxes. The sequences encoding the polymerase (Pol) I and II promoters, the Pol I promoter terminator (T), and the poly(A) tail (pA) are indicated. The position of restriction sites EcoRI and BsmBI, used to clone the NS1 sequences from patients 65 and 85, is shown. (B) Canine MDCK (left) and human A549 (right) cells were infected in duplicate with recombinant PR8 viruses expressing either 65 and 85 H3N2 NS1 proteins or the PR8 NS1 protein (wt-PR8-NSs) at an MOI of 0.001. Virus titers in infected cell supernatants were determined at different times postinfection by immunofocus assay. (C) MDCK cells were infected (MOI, 0.001) with viruses r65 and r85, and just after the infection, cells were left untreated or were treated with 2,000 U of IFN-α/well. Supernatants were collected at 24 and 48 hpi, and virus titers were determined by immunofocus assay. (Left) The percentage of growth in MDCK cells treated with IFN-α (+) was normalized to the growth in the nontreated cells (−) at 24 and 48 hpi (considered 100% growth). (Right) Virus titers (in FFU/ml) in nontreated (−) and IFN-treated (+) cells are shown. The experiments were repeated 3 times in duplicate wells. The dotted line in panel B indicates the limit of detection.

    Article Snippet: In the experiments to measure IFN sensitivity, the medium also contained 2,000 U/ml of universal IFN-α (Axxora).

    Techniques: Recombinant, In Vitro, Plasmid Preparation, Sequencing, Infection, Expressing

    Mutation I64T in IAV 1918 H1N1 NS1 affects its ability to inhibit general gene expression and innate immune responses. (A, B, and C) Human 293T cells were transiently cotransfected with pCAGGS plasmids expressing the different NS1 variants indicated and the WT 1918-NS1 (E26, I64, and R224), along with pCAGGS plasmids expressing the reporter proteins GFP and Gluc. (A) At 30 hpt, GFP expression was visualized using a fluorescence microscope. (B) Gluc expression was analyzed at 30 hpt using luminescence. Error bars represent the standard deviations for triplicates. P values using Student's t test are indicated. (C) In addition, NS1 and actin expression levels were analyzed by Western blotting from cell extracts using antibodies specific to the HA tag (to detect the NS1 protein) and to actin as a loading control. Western blots were quantified by densitometry using the software ImageJ (v1.46), and the amounts of NS1 protein were normalized to the amounts of actin protein. Protein expression in cells transfected with the PR8-NS1-expressing plasmid was considered to be 100% for comparison with the level of expression by the other NS1 variants (numbers below the NS1 blot). Molecular mass markers (in kilodaltons) are indicated on the right. Three different experiments were performed, with similar results. (D and E) Human A549 cells were transfected with pCAGGS plasmids expressing the 1918-NS1-I64, 1918-NS-T64, or PR8-NS1 protein using DNA-IN. At 24 hpt, cells were either transfected with 100 ng of poly(I·C) (D) or treated with 250 U/ml of universal IFN-α (E) to induce an antiviral cellular state. At 16 h posttreatment, cells were infected (MOI, 0.001) with VSV-GFP, and viral titers in the TCS were determined at 21 hpi. Bars represent the standard deviations from triplicates. Experiments were repeated 3 times in triplicate wells with similar results. P values using Student's t test are indicated.

    Journal: Journal of Virology

    Article Title: NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses

    doi: 10.1128/JVI.01039-16

    Figure Lengend Snippet: Mutation I64T in IAV 1918 H1N1 NS1 affects its ability to inhibit general gene expression and innate immune responses. (A, B, and C) Human 293T cells were transiently cotransfected with pCAGGS plasmids expressing the different NS1 variants indicated and the WT 1918-NS1 (E26, I64, and R224), along with pCAGGS plasmids expressing the reporter proteins GFP and Gluc. (A) At 30 hpt, GFP expression was visualized using a fluorescence microscope. (B) Gluc expression was analyzed at 30 hpt using luminescence. Error bars represent the standard deviations for triplicates. P values using Student's t test are indicated. (C) In addition, NS1 and actin expression levels were analyzed by Western blotting from cell extracts using antibodies specific to the HA tag (to detect the NS1 protein) and to actin as a loading control. Western blots were quantified by densitometry using the software ImageJ (v1.46), and the amounts of NS1 protein were normalized to the amounts of actin protein. Protein expression in cells transfected with the PR8-NS1-expressing plasmid was considered to be 100% for comparison with the level of expression by the other NS1 variants (numbers below the NS1 blot). Molecular mass markers (in kilodaltons) are indicated on the right. Three different experiments were performed, with similar results. (D and E) Human A549 cells were transfected with pCAGGS plasmids expressing the 1918-NS1-I64, 1918-NS-T64, or PR8-NS1 protein using DNA-IN. At 24 hpt, cells were either transfected with 100 ng of poly(I·C) (D) or treated with 250 U/ml of universal IFN-α (E) to induce an antiviral cellular state. At 16 h posttreatment, cells were infected (MOI, 0.001) with VSV-GFP, and viral titers in the TCS were determined at 21 hpi. Bars represent the standard deviations from triplicates. Experiments were repeated 3 times in triplicate wells with similar results. P values using Student's t test are indicated.

    Article Snippet: In the experiments to measure IFN sensitivity, the medium also contained 2,000 U/ml of universal IFN-α (Axxora).

    Techniques: Mutagenesis, Expressing, Fluorescence, Microscopy, Western Blot, Software, Transfection, Plasmid Preparation, Infection

    Resting levels of cellular proliferation and cell culture supernatant cytokines IFN-γ, IL-17, IL-10, IFN-α, and sCTLA-4 isolated from PBMC of SLE patients or age- and sex-matched healthy donors following incubation for 5 days ( n = 45 per group; * p

    Journal: Arthritis Research & Therapy

    Article Title: Immunoregulatory soluble CTLA-4 modifies effector T-cell responses in systemic lupus erythematosus

    doi: 10.1186/s13075-016-1075-1

    Figure Lengend Snippet: Resting levels of cellular proliferation and cell culture supernatant cytokines IFN-γ, IL-17, IL-10, IFN-α, and sCTLA-4 isolated from PBMC of SLE patients or age- and sex-matched healthy donors following incubation for 5 days ( n = 45 per group; * p

    Article Snippet: Cytokine standards (IL-10, IL-17, IFN-γ) were from Peprotech EC Ltd. (London, UK), and IFN-α was from Mabtech.

    Techniques: Cell Culture, Isolation, Incubation

    Specificity of IFN-α expressed by rhIFN-α BCG in regulating human peripheral blood mononuclear cell (PBMC) IFN-γ, IL-10 and IFN-inducible protein 10 (IP-10) production. PBMC were incubated with rhIFN-α BCG (rBCG, 0·01 OD 600 /ml) or rBCG (0·01 OD 600 /ml) plus neutralizing anti-IFN-α antibody (3 μg/ml) for 3 days. As controls, PBMC were incubated with medium, IFN-α (50 IU/ml), MV261 BCG (0·01 OD 600 /ml), or MV261 BCG (0·01 OD 600 /ml) plus IFN-α (50 IU/ml). IFN-γ, IL-10 and IP-10 in the cultures were quantified and the values were expressed as means ±s.d. from triplicate-well incubations.

    Journal: Clinical and Experimental Immunology

    Article Title: Recombinant bacille Calmette-Gu?rin (BCG) expressing human interferon-alpha 2B demonstrates enhanced immunogenicity

    doi: 10.1046/j.1365-2249.2001.01428.x

    Figure Lengend Snippet: Specificity of IFN-α expressed by rhIFN-α BCG in regulating human peripheral blood mononuclear cell (PBMC) IFN-γ, IL-10 and IFN-inducible protein 10 (IP-10) production. PBMC were incubated with rhIFN-α BCG (rBCG, 0·01 OD 600 /ml) or rBCG (0·01 OD 600 /ml) plus neutralizing anti-IFN-α antibody (3 μg/ml) for 3 days. As controls, PBMC were incubated with medium, IFN-α (50 IU/ml), MV261 BCG (0·01 OD 600 /ml), or MV261 BCG (0·01 OD 600 /ml) plus IFN-α (50 IU/ml). IFN-γ, IL-10 and IP-10 in the cultures were quantified and the values were expressed as means ±s.d. from triplicate-well incubations.

    Article Snippet: ELISA reagents including recombinant human cytokines and paired monoclonal capture and detecting antibodies for the cytokines were obtained from Endogen for IFN-α and -γ and from PharMingen (San Diego, CA) for IL-10 and IFN-inducible protein 10 (IP-10).

    Techniques: Incubation

    (a) Schematic illustration of the human IFN-α 2B-containing Escherichia coli –BCG shuttle plasmid. The IFN-α 2B coding sequence is placed downstream of BCG α-antigen signal sequence (SS) and influenza virus haemagglutinin epitope tag sequence (T). Expression of the cytokine is driven by the BCG heat shock protein (hsp)60 promoter (black box). The arrow indicates the direction of transcription. The kanamycin resistance cassette (open box) and a mycobacterial as well as an E. coli origin of replication are indicated. The illustration is not to scale. (b) Expression of IFN-α 2B in Western blot of BCG culture supernatant (S) and pellet lysate (P) from human IFN-α 2B BCG recombinant. Purified recombinant human IFN-α 2B is used as a positive control shown on the right. The blot is probed with a specific mouse anti-human IFN-α MoAb. Standard molecular weights are indicated.

    Journal: Clinical and Experimental Immunology

    Article Title: Recombinant bacille Calmette-Gu?rin (BCG) expressing human interferon-alpha 2B demonstrates enhanced immunogenicity

    doi: 10.1046/j.1365-2249.2001.01428.x

    Figure Lengend Snippet: (a) Schematic illustration of the human IFN-α 2B-containing Escherichia coli –BCG shuttle plasmid. The IFN-α 2B coding sequence is placed downstream of BCG α-antigen signal sequence (SS) and influenza virus haemagglutinin epitope tag sequence (T). Expression of the cytokine is driven by the BCG heat shock protein (hsp)60 promoter (black box). The arrow indicates the direction of transcription. The kanamycin resistance cassette (open box) and a mycobacterial as well as an E. coli origin of replication are indicated. The illustration is not to scale. (b) Expression of IFN-α 2B in Western blot of BCG culture supernatant (S) and pellet lysate (P) from human IFN-α 2B BCG recombinant. Purified recombinant human IFN-α 2B is used as a positive control shown on the right. The blot is probed with a specific mouse anti-human IFN-α MoAb. Standard molecular weights are indicated.

    Article Snippet: ELISA reagents including recombinant human cytokines and paired monoclonal capture and detecting antibodies for the cytokines were obtained from Endogen for IFN-α and -γ and from PharMingen (San Diego, CA) for IL-10 and IFN-inducible protein 10 (IP-10).

    Techniques: Plasmid Preparation, Sequencing, Expressing, Western Blot, Recombinant, Purification, Positive Control

    Maximum human peripheral blood mononuclear cell (PBMC) IFN-γ induction requires simultaneous presence of both BCG and IFN-α. PBMC were incubated with either MV261 BCG (0·01 OD 600 /ml) or IFN-α (100 IU/ml) for up to 4 h. At the indicated time points, the same doses of either IFN-α (for MV261 BCG prior cultured group) or MV261 BCG (for IFN-α prior cultured group) were added and the incubation continued for 3 days. IFN-γ in the cultures was quantified and the values were expressed as means ±s.d. from triplicate-well incubations.

    Journal: Clinical and Experimental Immunology

    Article Title: Recombinant bacille Calmette-Gu?rin (BCG) expressing human interferon-alpha 2B demonstrates enhanced immunogenicity

    doi: 10.1046/j.1365-2249.2001.01428.x

    Figure Lengend Snippet: Maximum human peripheral blood mononuclear cell (PBMC) IFN-γ induction requires simultaneous presence of both BCG and IFN-α. PBMC were incubated with either MV261 BCG (0·01 OD 600 /ml) or IFN-α (100 IU/ml) for up to 4 h. At the indicated time points, the same doses of either IFN-α (for MV261 BCG prior cultured group) or MV261 BCG (for IFN-α prior cultured group) were added and the incubation continued for 3 days. IFN-γ in the cultures was quantified and the values were expressed as means ±s.d. from triplicate-well incubations.

    Article Snippet: ELISA reagents including recombinant human cytokines and paired monoclonal capture and detecting antibodies for the cytokines were obtained from Endogen for IFN-α and -γ and from PharMingen (San Diego, CA) for IL-10 and IFN-inducible protein 10 (IP-10).

    Techniques: Incubation, Cell Culture