human lymphoblastoid cdna library Search Results


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  • 99
    New England Biolabs rrna depletion kit
    Rrna Depletion Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 644 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hela  (ATCC)
    99
    ATCC hela
    Critical contribution of RyDEN in IFN-mediated anti-DENV response. (A and B) Induction of RyDEN expression by IFN treatments. <t>HeLa</t> (A) and shRNA-expressing <t>HepG2</t> (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β mRNA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.
    Hela, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 11957 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Nugen ovation human ffpe rna seq kit
    Critical contribution of RyDEN in IFN-mediated anti-DENV response. (A and B) Induction of RyDEN expression by IFN treatments. <t>HeLa</t> (A) and shRNA-expressing <t>HepG2</t> (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β mRNA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.
    Ovation Human Ffpe Rna Seq Kit, supplied by Nugen, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher vector pcr3
    Critical contribution of RyDEN in IFN-mediated anti-DENV response. (A and B) Induction of RyDEN expression by IFN treatments. <t>HeLa</t> (A) and shRNA-expressing <t>HepG2</t> (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β mRNA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.
    Vector Pcr3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ion proton system
    Critical contribution of RyDEN in IFN-mediated anti-DENV response. (A and B) Induction of RyDEN expression by IFN treatments. <t>HeLa</t> (A) and shRNA-expressing <t>HepG2</t> (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β mRNA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.
    Ion Proton System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1523 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ion pi chip kit v3
    Critical contribution of RyDEN in IFN-mediated anti-DENV response. (A and B) Induction of RyDEN expression by IFN treatments. <t>HeLa</t> (A) and shRNA-expressing <t>HepG2</t> (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β mRNA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.
    Ion Pi Chip Kit V3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ion pi hi q sequencing 200 kit
    Critical contribution of RyDEN in IFN-mediated anti-DENV response. (A and B) Induction of RyDEN expression by IFN treatments. <t>HeLa</t> (A) and shRNA-expressing <t>HepG2</t> (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β mRNA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.
    Ion Pi Hi Q Sequencing 200 Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext ultra rna library prep kit for illumina
    Critical contribution of RyDEN in IFN-mediated anti-DENV response. (A and B) Induction of RyDEN expression by IFN treatments. <t>HeLa</t> (A) and shRNA-expressing <t>HepG2</t> (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β mRNA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.
    Nebnext Ultra Rna Library Prep Kit For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    5 PRIME rt pcr
    Critical contribution of RyDEN in IFN-mediated anti-DENV response. (A and B) Induction of RyDEN expression by IFN treatments. <t>HeLa</t> (A) and shRNA-expressing <t>HepG2</t> (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β mRNA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.
    Rt Pcr, supplied by 5 PRIME, 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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    Millipore ch7c17
    MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) <t>CH7C17</t> T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P
    Ch7c17, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher human hct 116 cells
    MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) <t>CH7C17</t> T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P
    Human Hct 116 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim expand high fidelity pcr
    MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) <t>CH7C17</t> T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P
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    Thermo Fisher flpin cho cells
    MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) <t>CH7C17</t> T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P
    Flpin Cho Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher flpin nih 3t3 cells
    MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) <t>CH7C17</t> T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P
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    Coriell Institute for Medical Research lymphoblastoid b cell lines
    MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) <t>CH7C17</t> T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P
    Lymphoblastoid B Cell Lines, supplied by Coriell Institute for Medical Research, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pef5frtv5gwcat vector
    MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) <t>CH7C17</t> T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P
    Pef5frtv5gwcat Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies quikchange site directed mutagenesis technique
    MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) <t>CH7C17</t> T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P
    Quikchange Site Directed Mutagenesis Technique, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) <t>CH7C17</t> T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P
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    MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) <t>CH7C17</t> T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P
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    MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) <t>CH7C17</t> T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P
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    MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) <t>CH7C17</t> T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P
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    Image Search Results


    Critical contribution of RyDEN in IFN-mediated anti-DENV response. (A and B) Induction of RyDEN expression by IFN treatments. HeLa (A) and shRNA-expressing HepG2 (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β mRNA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.

    Journal: PLoS Pathogens

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

    doi: 10.1371/journal.ppat.1005357

    Figure Lengend Snippet: Critical contribution of RyDEN in IFN-mediated anti-DENV response. (A and B) Induction of RyDEN expression by IFN treatments. HeLa (A) and shRNA-expressing HepG2 (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β mRNA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.

    Article Snippet: HeLa (cervical carcinoma), HepG2 (hepatoma), Huh7.5 (hepatocellular carcinoma), HEK293T (embryonic kidney), A549 (lung adenocarcinoma), Jurkat (lymphoblastoid T), and THP-1 (monocytic leukemia, ATCC TIB-202) cells were cultured in the presence (gray bars) or absence (white bars) of IFN-α/ω (1,000 U/ml).

    Techniques: Expressing, shRNA, Concentration Assay, Molecular Weight, Inhibition, Infection, Plaque Assay, Transfection, Plasmid Preparation, Isolation, Quantitative RT-PCR, Cell Culture

    Identification of RyDEN. (A) Procedure for gain-of-function screen. The cDNA library was generated from mRNA of IFN-α/ω-treated HeLa cells and transferred into a lentiviral vector by the Gateway recombination system. Infectious lentiviral vectors carrying the IFN cDNA library were produced as a VSV-G-pseudotyped virus and used to transduce DENV-susceptible Huh7.5 cells. cDNA library-expressing Huh7.5 cells were then challenged with DENV-2 at an MOI of 1, and cell colonies that survived DENV-induced cell death were collected. (B) Histogram analysis of cDNA fragments in library vectors. The entry vector (pDONR221, left panel) and destination vector (pYK005C, right panel) recombinated with the Gateway-compatible cDNA library were applied to Escherichia coli ( E . coli) , and the cells were spread onto LB plates to develop bacterial colonies. The cDNA fragments in individual colonies were amplified by PCR using primers described in Materials and Methods and visualized with agarose gel electrophoresis. The size of the PCR fragment was estimated by comparing the migration distance of the DNA molecular weight markers. Up to sixty colonies were picked up from each vector-transformed E . coli plate and analyzed. (C) Validation of DENV-resistant cell clones. Surviving clones obtained from (A) were seeded in a chamber slide and infected with DENV-2 at an MOI of 5. Two days after infection, cells were fixed with paraformaldehyde, permeabilized, and stained with anti-dsRNA antibody, followed by detection with Alexa Fluor 488-conjugated secondary antibody (red). Cell nuclei were stained with DAPI (blue). Representative merged images using four surviving clones (#1, 13, 14, and 15) and control cells (bacterial alkaline phosphatase [BAP]-expressing Huh7.5 cells) are shown. In a parallel experiment, the culture supernatant of infected cells was harvested 2 days after infection and subjected to plaque assay to measure the virus titer (insets). (D) Amplification of cDNA from DENV-resistant cells. Genomic DNA was isolated from cell clones, whose resistant property had been confirmed in (B), and cDNA was amplified by PCR using primers specific to the lentiviral vector. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (E) Amino acid sequence of RyDEN. (F) Predicted domain organization of RyDEN. RyDEN protein (291 amino acid) was suggested to contain eight α-helixes (blue), seven β-strands (orange), NLS (121–137), NES (261–269), zinc-ribbon domain (112–135), and coiled-coil motif (261–285). A unique glutamic acid-rich (E-rich) domain was also found in the C-terminus.

    Journal: PLoS Pathogens

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

    doi: 10.1371/journal.ppat.1005357

    Figure Lengend Snippet: Identification of RyDEN. (A) Procedure for gain-of-function screen. The cDNA library was generated from mRNA of IFN-α/ω-treated HeLa cells and transferred into a lentiviral vector by the Gateway recombination system. Infectious lentiviral vectors carrying the IFN cDNA library were produced as a VSV-G-pseudotyped virus and used to transduce DENV-susceptible Huh7.5 cells. cDNA library-expressing Huh7.5 cells were then challenged with DENV-2 at an MOI of 1, and cell colonies that survived DENV-induced cell death were collected. (B) Histogram analysis of cDNA fragments in library vectors. The entry vector (pDONR221, left panel) and destination vector (pYK005C, right panel) recombinated with the Gateway-compatible cDNA library were applied to Escherichia coli ( E . coli) , and the cells were spread onto LB plates to develop bacterial colonies. The cDNA fragments in individual colonies were amplified by PCR using primers described in Materials and Methods and visualized with agarose gel electrophoresis. The size of the PCR fragment was estimated by comparing the migration distance of the DNA molecular weight markers. Up to sixty colonies were picked up from each vector-transformed E . coli plate and analyzed. (C) Validation of DENV-resistant cell clones. Surviving clones obtained from (A) were seeded in a chamber slide and infected with DENV-2 at an MOI of 5. Two days after infection, cells were fixed with paraformaldehyde, permeabilized, and stained with anti-dsRNA antibody, followed by detection with Alexa Fluor 488-conjugated secondary antibody (red). Cell nuclei were stained with DAPI (blue). Representative merged images using four surviving clones (#1, 13, 14, and 15) and control cells (bacterial alkaline phosphatase [BAP]-expressing Huh7.5 cells) are shown. In a parallel experiment, the culture supernatant of infected cells was harvested 2 days after infection and subjected to plaque assay to measure the virus titer (insets). (D) Amplification of cDNA from DENV-resistant cells. Genomic DNA was isolated from cell clones, whose resistant property had been confirmed in (B), and cDNA was amplified by PCR using primers specific to the lentiviral vector. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (E) Amino acid sequence of RyDEN. (F) Predicted domain organization of RyDEN. RyDEN protein (291 amino acid) was suggested to contain eight α-helixes (blue), seven β-strands (orange), NLS (121–137), NES (261–269), zinc-ribbon domain (112–135), and coiled-coil motif (261–285). A unique glutamic acid-rich (E-rich) domain was also found in the C-terminus.

    Article Snippet: HeLa (cervical carcinoma), HepG2 (hepatoma), Huh7.5 (hepatocellular carcinoma), HEK293T (embryonic kidney), A549 (lung adenocarcinoma), Jurkat (lymphoblastoid T), and THP-1 (monocytic leukemia, ATCC TIB-202) cells were cultured in the presence (gray bars) or absence (white bars) of IFN-α/ω (1,000 U/ml).

    Techniques: cDNA Library Assay, Generated, Plasmid Preparation, Produced, Transduction, Expressing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Migration, Molecular Weight, Transformation Assay, Clone Assay, Infection, Staining, Plaque Assay, Isolation, Sequencing

    Inhibition of DENV replication by RyDEN expression. (A) Establishment of human cell lines stably expressing V5-tagged RyDEN. Huh7.5 and HepG2 cells expressing V5-RyDEN or V5-DHFR were created by lentiviral vector transduction and blasticidin selection. Expression of V5-tagged proteins was analyzed by immunoblotting (IB). Masses of molecular weight standards are indicated at left. (B) Replication of DENV-2 in stable cell lines. Huh7.5 (left panels) and HepG2 (right panels) cells expressing V5-RyDEN (gray) or V5-DHFR (white) were infected with DENV-2 (Singapore isolate) at MOIs of 0.1 (top), 1 (middle), and 10 (bottom), and virus replication was monitored until 72 h after infection. Infectious titers in culture supernatants were quantified by plaque assay. Note that, at an MOI of 10, the virus titer for V5-DHFR-expressing Huh7.5 cells peaked at the first day, and by the second day, a large proportion of the cells exhibited massive CPE, whereas V5-RyDEN-expressing Huh7.5 cells that displayed resistance to DENV-induced CPE produced steady level of viruses even after 2 days. (C) Inhibitory effect of RyDEN against all DENV serotypes. HepG2 cells expressing V5-RyDEN (gray bars) and V5-DHFR (white bars) were infected with DENV-1, -3, -4 (Singapore isolates), or -2 (New Guinea strain [NGC]) at an MOI of 0.1, and the virus titer was determined 2 days after infection. (D) shRNA-based knockdown of RyDEN mRNA. HeLa cells were transduced with lentiviral vectors expressing three different shRNA sequences against RyDEN mRNA (sh1425, sh3151, or sh5890) and subjected to puromycin selection to create stable cell lines. The expression level of RyDEN mRNA in RyDEN shRNA and non-targeting control shRNA (shCtrl)-expressing cells were analyzed by qRT-PCR analysis and normalized with GAPDH mRNA levels. (E) Replication efficiency of DENV in the knockdown cells. shRNA-expressing HeLa cells were infected with DENV-2 at an MOI of 1, and the viral titer in culture supernatant 2 days after infection was quantified by plaque assay. (F) Add-back of shRNA-resistant RyDEN in knockdown cells. Control shRNA (shCtrl, left lanes) and RyDEN shRNA (sh1425, right lanes)-expressing cells were established again using HepG2 cells. Cells were further transduced with lentiviral vectors expressing sh1425-susceptible wild-type (WT) or sh1425-resistant (1425 R ) V5-RyDEN and selected with blasticidin. The expression of V5-tagged RyDEN was analyzed by immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. Parental stands for the untransduced shRNA cell line. (G) Effect of the shRNA-resistant RyDEN expression on DENV replication in knockdown cells. Cell lines created in (F) were infected with DENV-2 at an MOI of 0.1, and the virus titer 2 days after infection was determined. The level of virus titer in the culture supernatants of WT and 1425 R cells relative to the parent cells (derived from each shRNA-expressing cells) is shown. Statistical significance was determined by two-way ANOVA (B), Student’s t test (C), or one-way ANOVA with Dunnett’s multiple comparison test (D, E, and G). ns, no significance (i.e., P > 0.05).

    Journal: PLoS Pathogens

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

    doi: 10.1371/journal.ppat.1005357

    Figure Lengend Snippet: Inhibition of DENV replication by RyDEN expression. (A) Establishment of human cell lines stably expressing V5-tagged RyDEN. Huh7.5 and HepG2 cells expressing V5-RyDEN or V5-DHFR were created by lentiviral vector transduction and blasticidin selection. Expression of V5-tagged proteins was analyzed by immunoblotting (IB). Masses of molecular weight standards are indicated at left. (B) Replication of DENV-2 in stable cell lines. Huh7.5 (left panels) and HepG2 (right panels) cells expressing V5-RyDEN (gray) or V5-DHFR (white) were infected with DENV-2 (Singapore isolate) at MOIs of 0.1 (top), 1 (middle), and 10 (bottom), and virus replication was monitored until 72 h after infection. Infectious titers in culture supernatants were quantified by plaque assay. Note that, at an MOI of 10, the virus titer for V5-DHFR-expressing Huh7.5 cells peaked at the first day, and by the second day, a large proportion of the cells exhibited massive CPE, whereas V5-RyDEN-expressing Huh7.5 cells that displayed resistance to DENV-induced CPE produced steady level of viruses even after 2 days. (C) Inhibitory effect of RyDEN against all DENV serotypes. HepG2 cells expressing V5-RyDEN (gray bars) and V5-DHFR (white bars) were infected with DENV-1, -3, -4 (Singapore isolates), or -2 (New Guinea strain [NGC]) at an MOI of 0.1, and the virus titer was determined 2 days after infection. (D) shRNA-based knockdown of RyDEN mRNA. HeLa cells were transduced with lentiviral vectors expressing three different shRNA sequences against RyDEN mRNA (sh1425, sh3151, or sh5890) and subjected to puromycin selection to create stable cell lines. The expression level of RyDEN mRNA in RyDEN shRNA and non-targeting control shRNA (shCtrl)-expressing cells were analyzed by qRT-PCR analysis and normalized with GAPDH mRNA levels. (E) Replication efficiency of DENV in the knockdown cells. shRNA-expressing HeLa cells were infected with DENV-2 at an MOI of 1, and the viral titer in culture supernatant 2 days after infection was quantified by plaque assay. (F) Add-back of shRNA-resistant RyDEN in knockdown cells. Control shRNA (shCtrl, left lanes) and RyDEN shRNA (sh1425, right lanes)-expressing cells were established again using HepG2 cells. Cells were further transduced with lentiviral vectors expressing sh1425-susceptible wild-type (WT) or sh1425-resistant (1425 R ) V5-RyDEN and selected with blasticidin. The expression of V5-tagged RyDEN was analyzed by immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. Parental stands for the untransduced shRNA cell line. (G) Effect of the shRNA-resistant RyDEN expression on DENV replication in knockdown cells. Cell lines created in (F) were infected with DENV-2 at an MOI of 0.1, and the virus titer 2 days after infection was determined. The level of virus titer in the culture supernatants of WT and 1425 R cells relative to the parent cells (derived from each shRNA-expressing cells) is shown. Statistical significance was determined by two-way ANOVA (B), Student’s t test (C), or one-way ANOVA with Dunnett’s multiple comparison test (D, E, and G). ns, no significance (i.e., P > 0.05).

    Article Snippet: HeLa (cervical carcinoma), HepG2 (hepatoma), Huh7.5 (hepatocellular carcinoma), HEK293T (embryonic kidney), A549 (lung adenocarcinoma), Jurkat (lymphoblastoid T), and THP-1 (monocytic leukemia, ATCC TIB-202) cells were cultured in the presence (gray bars) or absence (white bars) of IFN-α/ω (1,000 U/ml).

    Techniques: Inhibition, Expressing, Stable Transfection, Plasmid Preparation, Transduction, Selection, Molecular Weight, Infection, Plaque Assay, Produced, shRNA, Quantitative RT-PCR, Derivative Assay

    MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) CH7C17 T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P

    Journal: Nature Communications

    Article Title: Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells

    doi: 10.1038/ncomms1285

    Figure Lengend Snippet: MVBs in T cells translocate to the IS. ( a ) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. ( b ) CH7C17 T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b . ( c ) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. * P

    Article Snippet: 2 Vβ8) and CH7C17 (Vβ3 TCR specific for HA peptide) and the lymphoblastoid B-cell lines Raji (Burkitt lymphoma), HOM-2 (HLA-DR1 EBV-transformed) and BLS-1 (HLA class II-null B-LCL, generated from cells of patients with type II Bare lymphocyte syndrome) were cultured in RPMI (Sigma) containing 10% fetal bovine serum (Invitrogen).

    Techniques: Staining, Marker

    Exosomal miRNA-335 is transferred from T cell to APC in an Ag-specific manner. ( a ) Levels of miR-335 were assessed by quantitative reverse transcription PCR (qRT–PCR) in primary dendritic cells and T lymphoblasts, and in Raji and J77 cells. J77-CD63-GFP cells were stably transduced with miR-335 (J-335 cells) or miR-101 (J-101 cells), and miR-335 levels were determined by qRT–PCR in cells and derived exosomes. Data are representative of three experiments (mean and s.e.m.) ( b ) miR-335 levels in SEE-primed Raji cells sorted 24 h after conjugation with J-335 cells. J-101 were used as control donor cells. Left panel, Data are representative of seven independent experiments (mean and s.e.m), P =0.014 (one-sample t -test). Right panel, n =5 independent experiments; P =0.04 (one-sample t -test); error bars represent s.e.m. ( c ) miR-335 levels in HA-primed HOM-2 cells sorted 24 h after conjugation with CH7C17 cells overexpressing miR-335 (C-335). CH7C17 cells overexpressing miR-101 (C-101) were used as control donor cells. Data are representative of five experiments (mean and s.e.m.). ( d ) miR-335 and miR-92a levels in SEE-primed Raji cells sorted 24 h after conjugation with primary T lymphoblasts-expressing miR-335 and miR-92a endogenously. Data are representative of three experiments (mean and s.e.m.), * P =0.026 (one-sample t -test) AU, arbitrary units.

    Journal: Nature Communications

    Article Title: Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells

    doi: 10.1038/ncomms1285

    Figure Lengend Snippet: Exosomal miRNA-335 is transferred from T cell to APC in an Ag-specific manner. ( a ) Levels of miR-335 were assessed by quantitative reverse transcription PCR (qRT–PCR) in primary dendritic cells and T lymphoblasts, and in Raji and J77 cells. J77-CD63-GFP cells were stably transduced with miR-335 (J-335 cells) or miR-101 (J-101 cells), and miR-335 levels were determined by qRT–PCR in cells and derived exosomes. Data are representative of three experiments (mean and s.e.m.) ( b ) miR-335 levels in SEE-primed Raji cells sorted 24 h after conjugation with J-335 cells. J-101 were used as control donor cells. Left panel, Data are representative of seven independent experiments (mean and s.e.m), P =0.014 (one-sample t -test). Right panel, n =5 independent experiments; P =0.04 (one-sample t -test); error bars represent s.e.m. ( c ) miR-335 levels in HA-primed HOM-2 cells sorted 24 h after conjugation with CH7C17 cells overexpressing miR-335 (C-335). CH7C17 cells overexpressing miR-101 (C-101) were used as control donor cells. Data are representative of five experiments (mean and s.e.m.). ( d ) miR-335 and miR-92a levels in SEE-primed Raji cells sorted 24 h after conjugation with primary T lymphoblasts-expressing miR-335 and miR-92a endogenously. Data are representative of three experiments (mean and s.e.m.), * P =0.026 (one-sample t -test) AU, arbitrary units.

    Article Snippet: 2 Vβ8) and CH7C17 (Vβ3 TCR specific for HA peptide) and the lymphoblastoid B-cell lines Raji (Burkitt lymphoma), HOM-2 (HLA-DR1 EBV-transformed) and BLS-1 (HLA class II-null B-LCL, generated from cells of patients with type II Bare lymphocyte syndrome) were cultured in RPMI (Sigma) containing 10% fetal bovine serum (Invitrogen).

    Techniques: Polymerase Chain Reaction, Quantitative RT-PCR, Stable Transfection, Transduction, Derivative Assay, Conjugation Assay, Expressing