96 well plates Thermo Fisher Search Results


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  • 90
    Thermo Fisher nunc 96 well plate
    Nunc 96 Well Plate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nunc 96 well plate - by Bioz Stars, 2020-02
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    78
    Thermo Fisher frozen thermo fisher custom made 96 well microtiter plate
    Frozen Thermo Fisher Custom Made 96 Well Microtiter Plate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher 96 well deep well plates
    Polyribosome fractionation from HaCaT cells. Polysome profile of HaCaT cells. ( a ) HaCaT cell lysate was loaded on the top of centrifuge tubes, centrifuged at 26 000 r.p.m. at 4°C for 4 h. ( b ) HaCaT cell lysate was loaded on the top of sucrose gradient of <t>96-well</t> deep-well plate, centrifuged at 6000 r.p.m. at 4°C for 40 h. ( c ) HaCaT cell lysate, into which EDTA was added, was loaded on the top of sucrose gradient of 96-well deep-well plate, centrifuged at 6000 r.p.m. at 4°C for 40 h. Gradient bottom is to the left.
    96 Well Deep Well Plates, supplied by Thermo Fisher, 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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    90
    Thermo Fisher 96 well plates
    Inflammatory responses to VZV-infected HELF are specific to cells of human origin. (A) Monocytes were seeded at a density of 50,000 cells per well in <t>96-well</t> plates. VZV-infected or uninfected HELF were added to monocytes at a ratio of 1:1 or 1:10, or
    96 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1275 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher 96 well culture plate
    IRF1 is required for the expression of ISGs. ( A ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in <t>96-well</t> culture plates. Then, cells were transfected with increasing doses of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone. After 24 hours, luciferase expression was determined. Experiment was performed in triplicate, and data represent means ± SD. ( B ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in 12-well culture plates. Cells were transfected with ssRNA (20 ng/well), and incubated in the presence of DD264 (40 µM) or DMSO alone. After 24 h of culture, total RNAs were extracted, and expression levels of indicated genes were quantified by qRT-PCR. GAPDH , HPRT1 and 18S correspond to control housekeeping genes, whereas others are well-known ISGs. Data were normalized using cells stimulated with ssRNA alone as a reference (red bars).
    96 Well Culture Plate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 278 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher black 96 well immuno plates
    IRF1 is required for the expression of ISGs. ( A ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in <t>96-well</t> culture plates. Then, cells were transfected with increasing doses of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone. After 24 hours, luciferase expression was determined. Experiment was performed in triplicate, and data represent means ± SD. ( B ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in 12-well culture plates. Cells were transfected with ssRNA (20 ng/well), and incubated in the presence of DD264 (40 µM) or DMSO alone. After 24 h of culture, total RNAs were extracted, and expression levels of indicated genes were quantified by qRT-PCR. GAPDH , HPRT1 and 18S correspond to control housekeeping genes, whereas others are well-known ISGs. Data were normalized using cells stimulated with ssRNA alone as a reference (red bars).
    Black 96 Well Immuno Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher 96 well microdialysis plate
    IRF1 is required for the expression of ISGs. ( A ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in <t>96-well</t> culture plates. Then, cells were transfected with increasing doses of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone. After 24 hours, luciferase expression was determined. Experiment was performed in triplicate, and data represent means ± SD. ( B ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in 12-well culture plates. Cells were transfected with ssRNA (20 ng/well), and incubated in the presence of DD264 (40 µM) or DMSO alone. After 24 h of culture, total RNAs were extracted, and expression levels of indicated genes were quantified by qRT-PCR. GAPDH , HPRT1 and 18S correspond to control housekeeping genes, whereas others are well-known ISGs. Data were normalized using cells stimulated with ssRNA alone as a reference (red bars).
    96 Well Microdialysis Plate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher nunc fluoronunc luminunc 96 well plates
    IRF1 is required for the expression of ISGs. ( A ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in <t>96-well</t> culture plates. Then, cells were transfected with increasing doses of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone. After 24 hours, luciferase expression was determined. Experiment was performed in triplicate, and data represent means ± SD. ( B ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in 12-well culture plates. Cells were transfected with ssRNA (20 ng/well), and incubated in the presence of DD264 (40 µM) or DMSO alone. After 24 h of culture, total RNAs were extracted, and expression levels of indicated genes were quantified by qRT-PCR. GAPDH , HPRT1 and 18S correspond to control housekeeping genes, whereas others are well-known ISGs. Data were normalized using cells stimulated with ssRNA alone as a reference (red bars).
    Nunc Fluoronunc Luminunc 96 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher 96 well plate individual square well
    IRF1 is required for the expression of ISGs. ( A ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in <t>96-well</t> culture plates. Then, cells were transfected with increasing doses of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone. After 24 hours, luciferase expression was determined. Experiment was performed in triplicate, and data represent means ± SD. ( B ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in 12-well culture plates. Cells were transfected with ssRNA (20 ng/well), and incubated in the presence of DD264 (40 µM) or DMSO alone. After 24 h of culture, total RNAs were extracted, and expression levels of indicated genes were quantified by qRT-PCR. GAPDH , HPRT1 and 18S correspond to control housekeeping genes, whereas others are well-known ISGs. Data were normalized using cells stimulated with ssRNA alone as a reference (red bars).
    96 Well Plate Individual Square Well, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher 96 well plastic plate
    IRF1 is required for the expression of ISGs. ( A ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in <t>96-well</t> culture plates. Then, cells were transfected with increasing doses of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone. After 24 hours, luciferase expression was determined. Experiment was performed in triplicate, and data represent means ± SD. ( B ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in 12-well culture plates. Cells were transfected with ssRNA (20 ng/well), and incubated in the presence of DD264 (40 µM) or DMSO alone. After 24 h of culture, total RNAs were extracted, and expression levels of indicated genes were quantified by qRT-PCR. GAPDH , HPRT1 and 18S correspond to control housekeeping genes, whereas others are well-known ISGs. Data were normalized using cells stimulated with ssRNA alone as a reference (red bars).
    96 Well Plastic Plate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher 96 well polystyrene plates
    Roles of CLP proteins in bacterial aggregation. (A) Cells were grown in liquid LB medium for 72 h in <t>96-well</t> plates. The images were obtained by viewing from the top to the bottom. (B) Cells viewed from front to back after standing for 10 h following 24 h incubation in glass test tubes. (C) Cell sedimentation assy. WT, Δ clpA , Δ clpB , Δ clpC , and Δ clpD bacteria were grown until OD 600 = 0.7 and the bacterial precipitates were suspended by mixing, before the OD 600 values were measured at 1 h intervals.
    96 Well Polystyrene Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher nunc 96 well plates
    Roles of CLP proteins in bacterial aggregation. (A) Cells were grown in liquid LB medium for 72 h in <t>96-well</t> plates. The images were obtained by viewing from the top to the bottom. (B) Cells viewed from front to back after standing for 10 h following 24 h incubation in glass test tubes. (C) Cell sedimentation assy. WT, Δ clpA , Δ clpB , Δ clpC , and Δ clpD bacteria were grown until OD 600 = 0.7 and the bacterial precipitates were suspended by mixing, before the OD 600 values were measured at 1 h intervals.
    Nunc 96 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher plate septa
    Roles of CLP proteins in bacterial aggregation. (A) Cells were grown in liquid LB medium for 72 h in <t>96-well</t> plates. The images were obtained by viewing from the top to the bottom. (B) Cells viewed from front to back after standing for 10 h following 24 h incubation in glass test tubes. (C) Cell sedimentation assy. WT, Δ clpA , Δ clpB , Δ clpC , and Δ clpD bacteria were grown until OD 600 = 0.7 and the bacterial precipitates were suspended by mixing, before the OD 600 values were measured at 1 h intervals.
    Plate Septa, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher clear flat bottom immuno sterile 96 well plates
    Roles of CLP proteins in bacterial aggregation. (A) Cells were grown in liquid LB medium for 72 h in <t>96-well</t> plates. The images were obtained by viewing from the top to the bottom. (B) Cells viewed from front to back after standing for 10 h following 24 h incubation in glass test tubes. (C) Cell sedimentation assy. WT, Δ clpA , Δ clpB , Δ clpC , and Δ clpD bacteria were grown until OD 600 = 0.7 and the bacterial precipitates were suspended by mixing, before the OD 600 values were measured at 1 h intervals.
    Clear Flat Bottom Immuno Sterile 96 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher 96 well plate clear bottom
    Roles of CLP proteins in bacterial aggregation. (A) Cells were grown in liquid LB medium for 72 h in <t>96-well</t> plates. The images were obtained by viewing from the top to the bottom. (B) Cells viewed from front to back after standing for 10 h following 24 h incubation in glass test tubes. (C) Cell sedimentation assy. WT, Δ clpA , Δ clpB , Δ clpC , and Δ clpD bacteria were grown until OD 600 = 0.7 and the bacterial precipitates were suspended by mixing, before the OD 600 values were measured at 1 h intervals.
    96 Well Plate Clear Bottom, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher microamp optical 96
    Roles of CLP proteins in bacterial aggregation. (A) Cells were grown in liquid LB medium for 72 h in <t>96-well</t> plates. The images were obtained by viewing from the top to the bottom. (B) Cells viewed from front to back after standing for 10 h following 24 h incubation in glass test tubes. (C) Cell sedimentation assy. WT, Δ clpA , Δ clpB , Δ clpC , and Δ clpD bacteria were grown until OD 600 = 0.7 and the bacterial precipitates were suspended by mixing, before the OD 600 values were measured at 1 h intervals.
    Microamp Optical 96, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher nunc 96 well polypropylene microwell plates
    Roles of CLP proteins in bacterial aggregation. (A) Cells were grown in liquid LB medium for 72 h in <t>96-well</t> plates. The images were obtained by viewing from the top to the bottom. (B) Cells viewed from front to back after standing for 10 h following 24 h incubation in glass test tubes. (C) Cell sedimentation assy. WT, Δ clpA , Δ clpB , Δ clpC , and Δ clpD bacteria were grown until OD 600 = 0.7 and the bacterial precipitates were suspended by mixing, before the OD 600 values were measured at 1 h intervals.
    Nunc 96 Well Polypropylene Microwell Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher white 96 well immuno plates
    Roles of CLP proteins in bacterial aggregation. (A) Cells were grown in liquid LB medium for 72 h in <t>96-well</t> plates. The images were obtained by viewing from the top to the bottom. (B) Cells viewed from front to back after standing for 10 h following 24 h incubation in glass test tubes. (C) Cell sedimentation assy. WT, Δ clpA , Δ clpB , Δ clpC , and Δ clpD bacteria were grown until OD 600 = 0.7 and the bacterial precipitates were suspended by mixing, before the OD 600 values were measured at 1 h intervals.
    White 96 Well Immuno Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher 96 well round bottom plate
    Nonsecosteroidal VDR ligands are potent agonists in keratinocytes and PBMCs. ( A ) LY2108491 and LY2109866 are potent inhibitors of keratinocyte proliferation. KerTr cells plated in <t>96-well</t> plates were dosed with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 72 hours at 37°C before BrdU incorporation into DNA was analyzed as a measure of cell proliferation. Results (mean α SEM) of experiments performed in triplicate are shown. ( B ) Nonsecosteroidal VDR ligands are potent inducers of CYP24 gene expression in keratinocytes. TaqMan quantitative RT-PCR (Q-PCR) was performed on total RNA prepared from KerTr cells treated with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. Levels of GAPDH mRNA were measured in all the samples, and the results were normalized and presented as fold induction (α SEM) compared with normalized CYP24 levels in vehicle-treated cells. ( C ) Nonsecosteroidal VDR ligands are efficacious in TPA- and PHA-activated PBMCs. Primary cells isolated from donors were stimulated with TPA (100 ng/ml) and PHA (25 μl/ml) and treated with vehicle or 100 nM each of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. TaqMan Q-PCR was performed on RNA obtained from vehicle-treated or VDR ligand–treated samples, using primer pairs and probes for IL-2, IL-4, IL-10, GATA3, and GAPDH. The amount of IL-2, IL-4, IL-10, and GATA3 transcripts relative to GAPDH transcripts is shown as mean α SEM of quadruplicate experimes.
    96 Well Round Bottom Plate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher clear flat bottom immuno nonsterile 96 well plates
    Nonsecosteroidal VDR ligands are potent agonists in keratinocytes and PBMCs. ( A ) LY2108491 and LY2109866 are potent inhibitors of keratinocyte proliferation. KerTr cells plated in <t>96-well</t> plates were dosed with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 72 hours at 37°C before BrdU incorporation into DNA was analyzed as a measure of cell proliferation. Results (mean α SEM) of experiments performed in triplicate are shown. ( B ) Nonsecosteroidal VDR ligands are potent inducers of CYP24 gene expression in keratinocytes. TaqMan quantitative RT-PCR (Q-PCR) was performed on total RNA prepared from KerTr cells treated with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. Levels of GAPDH mRNA were measured in all the samples, and the results were normalized and presented as fold induction (α SEM) compared with normalized CYP24 levels in vehicle-treated cells. ( C ) Nonsecosteroidal VDR ligands are efficacious in TPA- and PHA-activated PBMCs. Primary cells isolated from donors were stimulated with TPA (100 ng/ml) and PHA (25 μl/ml) and treated with vehicle or 100 nM each of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. TaqMan Q-PCR was performed on RNA obtained from vehicle-treated or VDR ligand–treated samples, using primer pairs and probes for IL-2, IL-4, IL-10, GATA3, and GAPDH. The amount of IL-2, IL-4, IL-10, and GATA3 transcripts relative to GAPDH transcripts is shown as mean α SEM of quadruplicate experimes.
    Clear Flat Bottom Immuno Nonsterile 96 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 434 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher nunc microwell 96 well optical bottom plates
    Nonsecosteroidal VDR ligands are potent agonists in keratinocytes and PBMCs. ( A ) LY2108491 and LY2109866 are potent inhibitors of keratinocyte proliferation. KerTr cells plated in <t>96-well</t> plates were dosed with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 72 hours at 37°C before BrdU incorporation into DNA was analyzed as a measure of cell proliferation. Results (mean α SEM) of experiments performed in triplicate are shown. ( B ) Nonsecosteroidal VDR ligands are potent inducers of CYP24 gene expression in keratinocytes. TaqMan quantitative RT-PCR (Q-PCR) was performed on total RNA prepared from KerTr cells treated with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. Levels of GAPDH mRNA were measured in all the samples, and the results were normalized and presented as fold induction (α SEM) compared with normalized CYP24 levels in vehicle-treated cells. ( C ) Nonsecosteroidal VDR ligands are efficacious in TPA- and PHA-activated PBMCs. Primary cells isolated from donors were stimulated with TPA (100 ng/ml) and PHA (25 μl/ml) and treated with vehicle or 100 nM each of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. TaqMan Q-PCR was performed on RNA obtained from vehicle-treated or VDR ligand–treated samples, using primer pairs and probes for IL-2, IL-4, IL-10, GATA3, and GAPDH. The amount of IL-2, IL-4, IL-10, and GATA3 transcripts relative to GAPDH transcripts is shown as mean α SEM of quadruplicate experimes.
    Nunc Microwell 96 Well Optical Bottom Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher round bottom 96 microwell
    Nonsecosteroidal VDR ligands are potent agonists in keratinocytes and PBMCs. ( A ) LY2108491 and LY2109866 are potent inhibitors of keratinocyte proliferation. KerTr cells plated in <t>96-well</t> plates were dosed with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 72 hours at 37°C before BrdU incorporation into DNA was analyzed as a measure of cell proliferation. Results (mean α SEM) of experiments performed in triplicate are shown. ( B ) Nonsecosteroidal VDR ligands are potent inducers of CYP24 gene expression in keratinocytes. TaqMan quantitative RT-PCR (Q-PCR) was performed on total RNA prepared from KerTr cells treated with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. Levels of GAPDH mRNA were measured in all the samples, and the results were normalized and presented as fold induction (α SEM) compared with normalized CYP24 levels in vehicle-treated cells. ( C ) Nonsecosteroidal VDR ligands are efficacious in TPA- and PHA-activated PBMCs. Primary cells isolated from donors were stimulated with TPA (100 ng/ml) and PHA (25 μl/ml) and treated with vehicle or 100 nM each of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. TaqMan Q-PCR was performed on RNA obtained from vehicle-treated or VDR ligand–treated samples, using primer pairs and probes for IL-2, IL-4, IL-10, GATA3, and GAPDH. The amount of IL-2, IL-4, IL-10, and GATA3 transcripts relative to GAPDH transcripts is shown as mean α SEM of quadruplicate experimes.
    Round Bottom 96 Microwell, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher 96 well plate cell harvester
    Nonsecosteroidal VDR ligands are potent agonists in keratinocytes and PBMCs. ( A ) LY2108491 and LY2109866 are potent inhibitors of keratinocyte proliferation. KerTr cells plated in <t>96-well</t> plates were dosed with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 72 hours at 37°C before BrdU incorporation into DNA was analyzed as a measure of cell proliferation. Results (mean α SEM) of experiments performed in triplicate are shown. ( B ) Nonsecosteroidal VDR ligands are potent inducers of CYP24 gene expression in keratinocytes. TaqMan quantitative RT-PCR (Q-PCR) was performed on total RNA prepared from KerTr cells treated with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. Levels of GAPDH mRNA were measured in all the samples, and the results were normalized and presented as fold induction (α SEM) compared with normalized CYP24 levels in vehicle-treated cells. ( C ) Nonsecosteroidal VDR ligands are efficacious in TPA- and PHA-activated PBMCs. Primary cells isolated from donors were stimulated with TPA (100 ng/ml) and PHA (25 μl/ml) and treated with vehicle or 100 nM each of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. TaqMan Q-PCR was performed on RNA obtained from vehicle-treated or VDR ligand–treated samples, using primer pairs and probes for IL-2, IL-4, IL-10, GATA3, and GAPDH. The amount of IL-2, IL-4, IL-10, and GATA3 transcripts relative to GAPDH transcripts is shown as mean α SEM of quadruplicate experimes.
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    Thermo Fisher clear c shaped immuno nonsterile 96 well plates
    Nonsecosteroidal VDR ligands are potent agonists in keratinocytes and PBMCs. ( A ) LY2108491 and LY2109866 are potent inhibitors of keratinocyte proliferation. KerTr cells plated in <t>96-well</t> plates were dosed with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 72 hours at 37°C before BrdU incorporation into DNA was analyzed as a measure of cell proliferation. Results (mean α SEM) of experiments performed in triplicate are shown. ( B ) Nonsecosteroidal VDR ligands are potent inducers of CYP24 gene expression in keratinocytes. TaqMan quantitative RT-PCR (Q-PCR) was performed on total RNA prepared from KerTr cells treated with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. Levels of GAPDH mRNA were measured in all the samples, and the results were normalized and presented as fold induction (α SEM) compared with normalized CYP24 levels in vehicle-treated cells. ( C ) Nonsecosteroidal VDR ligands are efficacious in TPA- and PHA-activated PBMCs. Primary cells isolated from donors were stimulated with TPA (100 ng/ml) and PHA (25 μl/ml) and treated with vehicle or 100 nM each of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. TaqMan Q-PCR was performed on RNA obtained from vehicle-treated or VDR ligand–treated samples, using primer pairs and probes for IL-2, IL-4, IL-10, GATA3, and GAPDH. The amount of IL-2, IL-4, IL-10, and GATA3 transcripts relative to GAPDH transcripts is shown as mean α SEM of quadruplicate experimes.
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    Thermo Fisher 96 well immulon plates
    Binding of wild-type α1 I domain to Toolkits II and III and to selected shorter triple-helical peptides. The recombinant wild-type α1 I domain was incubated in wells of <t>Immulon</t> 2HB 96-well plates coated with peptides, and adhesion was measured in the presence of either 2 m m Mg 2+ ( black bars ) or 2 m m EDTA ( white bars ) as described under “Experimental Procedures.” BSA, GPP10, and GFOGER served as control surface coatings. Experiments were performed in triplicate using 0.1 μg of protein/well. a , adhesion to Toolkit II; b , adhesion to Toolkit III; c , adhesion to selected shorter peptides, formatted with the indicated sequence flanked by (GPP) 5 host peptides. Long GFPGER has the same sequence as II-28 but with “O” in its GFOGER motif replaced with “P.” Data are the mean ± S.E. of at least six independent experiments. Col , collagen.
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    Thermo Fisher 96 well spe plate
    Binding of wild-type α1 I domain to Toolkits II and III and to selected shorter triple-helical peptides. The recombinant wild-type α1 I domain was incubated in wells of <t>Immulon</t> 2HB 96-well plates coated with peptides, and adhesion was measured in the presence of either 2 m m Mg 2+ ( black bars ) or 2 m m EDTA ( white bars ) as described under “Experimental Procedures.” BSA, GPP10, and GFOGER served as control surface coatings. Experiments were performed in triplicate using 0.1 μg of protein/well. a , adhesion to Toolkit II; b , adhesion to Toolkit III; c , adhesion to selected shorter peptides, formatted with the indicated sequence flanked by (GPP) 5 host peptides. Long GFPGER has the same sequence as II-28 but with “O” in its GFOGER motif replaced with “P.” Data are the mean ± S.E. of at least six independent experiments. Col , collagen.
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    Thermo Fisher hiv 1 rt
    Crystal structures of LEDGF/IBD (A), BI-1001 (B), KF115 (C), and KF116 (D) bound to <t>HIV-1</t> IN CCD. The IN subunit 1 and 2 are colored in cyan and green, respectively. LEDGF/IBD loop (amino acids 365–368) is shown in dark blue. BI-1001 is shown in orange. KF115 is shown in red. KF116 is shown in magenta. The hydrogen bonds between the IN subunit and the LEDGF/IBD or the indicated inhibitors are shown by black dashed lines. Side chains of HIV-1 IN residues A128T and T125 in subunit 1, and E170, H171 and T174 in subunit 2 are shown.
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    Thermo Fisher nunc maxisorp 96 well plate
    Crystal structures of LEDGF/IBD (A), BI-1001 (B), KF115 (C), and KF116 (D) bound to <t>HIV-1</t> IN CCD. The IN subunit 1 and 2 are colored in cyan and green, respectively. LEDGF/IBD loop (amino acids 365–368) is shown in dark blue. BI-1001 is shown in orange. KF115 is shown in red. KF116 is shown in magenta. The hydrogen bonds between the IN subunit and the LEDGF/IBD or the indicated inhibitors are shown by black dashed lines. Side chains of HIV-1 IN residues A128T and T125 in subunit 1, and E170, H171 and T174 in subunit 2 are shown.
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    Thermo Fisher microamp 96 well full plate cover
    Crystal structures of LEDGF/IBD (A), BI-1001 (B), KF115 (C), and KF116 (D) bound to <t>HIV-1</t> IN CCD. The IN subunit 1 and 2 are colored in cyan and green, respectively. LEDGF/IBD loop (amino acids 365–368) is shown in dark blue. BI-1001 is shown in orange. KF115 is shown in red. KF116 is shown in magenta. The hydrogen bonds between the IN subunit and the LEDGF/IBD or the indicated inhibitors are shown by black dashed lines. Side chains of HIV-1 IN residues A128T and T125 in subunit 1, and E170, H171 and T174 in subunit 2 are shown.
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    Thermo Fisher 96 well nunc f96 microwell plates
    Crystal structures of LEDGF/IBD (A), BI-1001 (B), KF115 (C), and KF116 (D) bound to <t>HIV-1</t> IN CCD. The IN subunit 1 and 2 are colored in cyan and green, respectively. LEDGF/IBD loop (amino acids 365–368) is shown in dark blue. BI-1001 is shown in orange. KF115 is shown in red. KF116 is shown in magenta. The hydrogen bonds between the IN subunit and the LEDGF/IBD or the indicated inhibitors are shown by black dashed lines. Side chains of HIV-1 IN residues A128T and T125 in subunit 1, and E170, H171 and T174 in subunit 2 are shown.
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    Thermo Fisher 96 well maxisorp plates
    Crystal structures of LEDGF/IBD (A), BI-1001 (B), KF115 (C), and KF116 (D) bound to <t>HIV-1</t> IN CCD. The IN subunit 1 and 2 are colored in cyan and green, respectively. LEDGF/IBD loop (amino acids 365–368) is shown in dark blue. BI-1001 is shown in orange. KF115 is shown in red. KF116 is shown in magenta. The hydrogen bonds between the IN subunit and the LEDGF/IBD or the indicated inhibitors are shown by black dashed lines. Side chains of HIV-1 IN residues A128T and T125 in subunit 1, and E170, H171 and T174 in subunit 2 are shown.
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    Image Search Results


    Polyribosome fractionation from HaCaT cells. Polysome profile of HaCaT cells. ( a ) HaCaT cell lysate was loaded on the top of centrifuge tubes, centrifuged at 26 000 r.p.m. at 4°C for 4 h. ( b ) HaCaT cell lysate was loaded on the top of sucrose gradient of 96-well deep-well plate, centrifuged at 6000 r.p.m. at 4°C for 40 h. ( c ) HaCaT cell lysate, into which EDTA was added, was loaded on the top of sucrose gradient of 96-well deep-well plate, centrifuged at 6000 r.p.m. at 4°C for 40 h. Gradient bottom is to the left.

    Journal: Nucleic Acids Research

    Article Title: High-throughput polyribosome fractionation

    doi: 10.1093/nar/gnh077

    Figure Lengend Snippet: Polyribosome fractionation from HaCaT cells. Polysome profile of HaCaT cells. ( a ) HaCaT cell lysate was loaded on the top of centrifuge tubes, centrifuged at 26 000 r.p.m. at 4°C for 4 h. ( b ) HaCaT cell lysate was loaded on the top of sucrose gradient of 96-well deep-well plate, centrifuged at 6000 r.p.m. at 4°C for 40 h. ( c ) HaCaT cell lysate, into which EDTA was added, was loaded on the top of sucrose gradient of 96-well deep-well plate, centrifuged at 6000 r.p.m. at 4°C for 40 h. Gradient bottom is to the left.

    Article Snippet: 96-well Deep Well plates (Nalge Nunc International, Cat. No. 270141) were used for sucrose gradients.

    Techniques: Fractionation

    Inflammatory responses to VZV-infected HELF are specific to cells of human origin. (A) Monocytes were seeded at a density of 50,000 cells per well in 96-well plates. VZV-infected or uninfected HELF were added to monocytes at a ratio of 1:1 or 1:10, or

    Journal:

    Article Title: Varicella-Zoster Virus Activates Inflammatory Cytokines in Human Monocytes and Macrophages via Toll-Like Receptor 2

    doi: 10.1128/JVI.79.20.12658-12666.2005

    Figure Lengend Snippet: Inflammatory responses to VZV-infected HELF are specific to cells of human origin. (A) Monocytes were seeded at a density of 50,000 cells per well in 96-well plates. VZV-infected or uninfected HELF were added to monocytes at a ratio of 1:1 or 1:10, or

    Article Snippet: Monocytes were seeded into 96-well plates at a density of 5 × 104 cells/well or in 24-well plates at 5 × 105 cells/well in RPMI (Gibco) with 18% autologous serum.

    Techniques: Infection

    IRF1 is required for the expression of ISGs. ( A ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in 96-well culture plates. Then, cells were transfected with increasing doses of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone. After 24 hours, luciferase expression was determined. Experiment was performed in triplicate, and data represent means ± SD. ( B ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in 12-well culture plates. Cells were transfected with ssRNA (20 ng/well), and incubated in the presence of DD264 (40 µM) or DMSO alone. After 24 h of culture, total RNAs were extracted, and expression levels of indicated genes were quantified by qRT-PCR. GAPDH , HPRT1 and 18S correspond to control housekeeping genes, whereas others are well-known ISGs. Data were normalized using cells stimulated with ssRNA alone as a reference (red bars).

    Journal: PLoS Pathogens

    Article Title: Inhibition of Pyrimidine Biosynthesis Pathway Suppresses Viral Growth through Innate Immunity

    doi: 10.1371/journal.ppat.1003678

    Figure Lengend Snippet: IRF1 is required for the expression of ISGs. ( A ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in 96-well culture plates. Then, cells were transfected with increasing doses of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone. After 24 hours, luciferase expression was determined. Experiment was performed in triplicate, and data represent means ± SD. ( B ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against IRF1 and cultured for 48 hours in 12-well culture plates. Cells were transfected with ssRNA (20 ng/well), and incubated in the presence of DD264 (40 µM) or DMSO alone. After 24 h of culture, total RNAs were extracted, and expression levels of indicated genes were quantified by qRT-PCR. GAPDH , HPRT1 and 18S correspond to control housekeeping genes, whereas others are well-known ISGs. Data were normalized using cells stimulated with ssRNA alone as a reference (red bars).

    Article Snippet: For one 96-well culture plate, 17 µg of plasmid were diluted in 500 µl of DMEM (Gibco-Invitrogen).

    Techniques: Expressing, Luciferase, Transfection, Cell Culture, Incubation, Quantitative RT-PCR

    DD264 activates the ISRE-luciferase reporter gene. ( A ) Bar chart showing results for one 96-well screening plate. ISRE-luciferase reporter gene induction is expressed as a fold change compared to control wells treated with DMSO alone. Each bar represents one compound and the red bar corresponds to DD264 (induction factor = 6.9). Compounds from this representative plate were from Institut Curie library and were screened at 20 µg/ml, thus corresponding to a final concentration of 81.1 µM for DD264. ( B ) Chemical structure of DD264. ( C ) HEK-293 cells that express luciferase under control of five interferon-stimulated response elements (ISRE) were incubated with increasing doses of DD264 or DMSO alone. After 24 hours, luciferase expression was determined. ( D ) Same experiment as in (C), but cells were stimulated with increasing doses of recombinant IFN-β. Except for (A) that corresponds to one representative experiment, all experiments were performed in triplicate, and data represent means ± SD.

    Journal: PLoS Pathogens

    Article Title: Inhibition of Pyrimidine Biosynthesis Pathway Suppresses Viral Growth through Innate Immunity

    doi: 10.1371/journal.ppat.1003678

    Figure Lengend Snippet: DD264 activates the ISRE-luciferase reporter gene. ( A ) Bar chart showing results for one 96-well screening plate. ISRE-luciferase reporter gene induction is expressed as a fold change compared to control wells treated with DMSO alone. Each bar represents one compound and the red bar corresponds to DD264 (induction factor = 6.9). Compounds from this representative plate were from Institut Curie library and were screened at 20 µg/ml, thus corresponding to a final concentration of 81.1 µM for DD264. ( B ) Chemical structure of DD264. ( C ) HEK-293 cells that express luciferase under control of five interferon-stimulated response elements (ISRE) were incubated with increasing doses of DD264 or DMSO alone. After 24 hours, luciferase expression was determined. ( D ) Same experiment as in (C), but cells were stimulated with increasing doses of recombinant IFN-β. Except for (A) that corresponds to one representative experiment, all experiments were performed in triplicate, and data represent means ± SD.

    Article Snippet: For one 96-well culture plate, 17 µg of plasmid were diluted in 500 µl of DMEM (Gibco-Invitrogen).

    Techniques: Luciferase, Concentration Assay, Incubation, Expressing, Recombinant

    Inhibition of pyrimidine biosynthesis accounts for the amplified cellular response to ssRNA. ( A ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with increasing doses of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone in 96-well cultures plates. Culture medium was supplemented or not with uridine. After 24 hours, luciferase expression was determined. Experiment was performed in triplicate, and data represent means ± SD. ( B ) Cells were transfected with 20 ng/well of ssRNA in 12-well plates, and incubated in the presence of DD264 (80 µM) or DMSO alone. Culture medium was supplemented or not with uridine. After 24 hours, expression levels of IFI6, IFI27, IFIT1 and IFI35 were determined by qRT-PCR. Data were normalized relative to control housekeeping genes (GAPDH, HPRT1, and 18S). Experiment was performed in duplicate, and data represent means ± SD. ( C ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected in 96-well plates with 50 ng of an expression vector encoding for DHODH (using two independent plasmid preparations of the same construct, #1 and #2). DHODH overexpression was assessed by western-blot analysis (upper right panel). Alternatively, cells were transfected with expression vectors encoding for control proteins (CT1 and CT2, see Materials and Methods for details). After 48 hours, cells were transfected with 6 ng/well of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone. After 24 hours, luciferase expression was determined. Experiment was performed in duplicate, and data represent means ± SD. ( D ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with increasing doses of ssRNA, and incubated in the presence of brequinar (200 nM) or DMSO alone in 96-well cultures plates. After 24 hours, luciferase expression was determined. Experiment was performed in duplicate, and data represent means ± SD.

    Journal: PLoS Pathogens

    Article Title: Inhibition of Pyrimidine Biosynthesis Pathway Suppresses Viral Growth through Innate Immunity

    doi: 10.1371/journal.ppat.1003678

    Figure Lengend Snippet: Inhibition of pyrimidine biosynthesis accounts for the amplified cellular response to ssRNA. ( A ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with increasing doses of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone in 96-well cultures plates. Culture medium was supplemented or not with uridine. After 24 hours, luciferase expression was determined. Experiment was performed in triplicate, and data represent means ± SD. ( B ) Cells were transfected with 20 ng/well of ssRNA in 12-well plates, and incubated in the presence of DD264 (80 µM) or DMSO alone. Culture medium was supplemented or not with uridine. After 24 hours, expression levels of IFI6, IFI27, IFIT1 and IFI35 were determined by qRT-PCR. Data were normalized relative to control housekeeping genes (GAPDH, HPRT1, and 18S). Experiment was performed in duplicate, and data represent means ± SD. ( C ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected in 96-well plates with 50 ng of an expression vector encoding for DHODH (using two independent plasmid preparations of the same construct, #1 and #2). DHODH overexpression was assessed by western-blot analysis (upper right panel). Alternatively, cells were transfected with expression vectors encoding for control proteins (CT1 and CT2, see Materials and Methods for details). After 48 hours, cells were transfected with 6 ng/well of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone. After 24 hours, luciferase expression was determined. Experiment was performed in duplicate, and data represent means ± SD. ( D ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with increasing doses of ssRNA, and incubated in the presence of brequinar (200 nM) or DMSO alone in 96-well cultures plates. After 24 hours, luciferase expression was determined. Experiment was performed in duplicate, and data represent means ± SD.

    Article Snippet: For one 96-well culture plate, 17 µg of plasmid were diluted in 500 µl of DMEM (Gibco-Invitrogen).

    Techniques: Inhibition, Amplification, Luciferase, Transfection, Incubation, Expressing, Quantitative RT-PCR, Plasmid Preparation, Construct, Over Expression, Western Blot

    DD264 amplifies cellular response to transfection of PAMP-like ssRNA molecules or IFN-β stimulation, and this correlates with its antiviral activity. ( A ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with increasing doses of synthetic 5′-triphosphate RNA molecules (ssRNA), and incubated in the presence of DD264 (80 µM) or DMSO alone in 96-well cultures plates. After 24 hours, luciferase expression was determined. ( B ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with increasing doses of recombinant IFN-β, and incubated in the presence of DD264 (80 µM) or DMSO alone in 96-well culture plates. After 24 hours, luciferase expression was determined. Experiments (A) and (B) were performed in duplicate, and data represent means ± SD. ( C ) Eight molecules were randomly picked among analogs of DD264 (see Table S2 ) to build a set of compounds with a range of antiviral activities from null to high as determined by their potency to inhibit MV-Luc replication. Then, the selected molecules were tested for the amplification ISRE-luciferase expression when stimulating cells with suboptimal doses of ssRNA (6 ng/well) as described in (A). Finally, for DD264 and selected analogs, the capacity to amplify cellular response to ssRNA was plotted as a function of the antiviral activity by means of the experimental IC 50 values (antiviral activity = 1/IC 50 *100).

    Journal: PLoS Pathogens

    Article Title: Inhibition of Pyrimidine Biosynthesis Pathway Suppresses Viral Growth through Innate Immunity

    doi: 10.1371/journal.ppat.1003678

    Figure Lengend Snippet: DD264 amplifies cellular response to transfection of PAMP-like ssRNA molecules or IFN-β stimulation, and this correlates with its antiviral activity. ( A ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with increasing doses of synthetic 5′-triphosphate RNA molecules (ssRNA), and incubated in the presence of DD264 (80 µM) or DMSO alone in 96-well cultures plates. After 24 hours, luciferase expression was determined. ( B ) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with increasing doses of recombinant IFN-β, and incubated in the presence of DD264 (80 µM) or DMSO alone in 96-well culture plates. After 24 hours, luciferase expression was determined. Experiments (A) and (B) were performed in duplicate, and data represent means ± SD. ( C ) Eight molecules were randomly picked among analogs of DD264 (see Table S2 ) to build a set of compounds with a range of antiviral activities from null to high as determined by their potency to inhibit MV-Luc replication. Then, the selected molecules were tested for the amplification ISRE-luciferase expression when stimulating cells with suboptimal doses of ssRNA (6 ng/well) as described in (A). Finally, for DD264 and selected analogs, the capacity to amplify cellular response to ssRNA was plotted as a function of the antiviral activity by means of the experimental IC 50 values (antiviral activity = 1/IC 50 *100).

    Article Snippet: For one 96-well culture plate, 17 µg of plasmid were diluted in 500 µl of DMEM (Gibco-Invitrogen).

    Techniques: Transfection, Activity Assay, Luciferase, Incubation, Expressing, Recombinant, Amplification

    Roles of CLP proteins in bacterial aggregation. (A) Cells were grown in liquid LB medium for 72 h in 96-well plates. The images were obtained by viewing from the top to the bottom. (B) Cells viewed from front to back after standing for 10 h following 24 h incubation in glass test tubes. (C) Cell sedimentation assy. WT, Δ clpA , Δ clpB , Δ clpC , and Δ clpD bacteria were grown until OD 600 = 0.7 and the bacterial precipitates were suspended by mixing, before the OD 600 values were measured at 1 h intervals.

    Journal: PLoS ONE

    Article Title: Collagen-Like Proteins (ClpA, ClpB, ClpC, and ClpD) Are Required for Biofilm Formation and Adhesion to Plant Roots by Bacillus amyloliquefaciens FZB42

    doi: 10.1371/journal.pone.0117414

    Figure Lengend Snippet: Roles of CLP proteins in bacterial aggregation. (A) Cells were grown in liquid LB medium for 72 h in 96-well plates. The images were obtained by viewing from the top to the bottom. (B) Cells viewed from front to back after standing for 10 h following 24 h incubation in glass test tubes. (C) Cell sedimentation assy. WT, Δ clpA , Δ clpB , Δ clpC , and Δ clpD bacteria were grown until OD 600 = 0.7 and the bacterial precipitates were suspended by mixing, before the OD 600 values were measured at 1 h intervals.

    Article Snippet: To quantify biofilm growth, we applied the crystal violet staining method in 96-well polystyrene plates (Thermo) [ – ].

    Techniques: Incubation, Sedimentation

    Variations in biofilm formation by the wild type and clp mutants. Biofilm formation by wild type and clp mutants in LB medium (A) and on MSgg medium plates (B). The images of colonies were obtained after incubation for 48 h at 37°C. (C) The biofilm images are top-down views of 96-well plates, which were obtained after incubation for 24 h at 37°C in MSgg liquid medium. (D) Quantitative spectrophotometric biofilm assay following crystal violet staining in MSgg medium. Analysis of variance detected a significant main group effect between the wild type and clp mutants (b, P

    Journal: PLoS ONE

    Article Title: Collagen-Like Proteins (ClpA, ClpB, ClpC, and ClpD) Are Required for Biofilm Formation and Adhesion to Plant Roots by Bacillus amyloliquefaciens FZB42

    doi: 10.1371/journal.pone.0117414

    Figure Lengend Snippet: Variations in biofilm formation by the wild type and clp mutants. Biofilm formation by wild type and clp mutants in LB medium (A) and on MSgg medium plates (B). The images of colonies were obtained after incubation for 48 h at 37°C. (C) The biofilm images are top-down views of 96-well plates, which were obtained after incubation for 24 h at 37°C in MSgg liquid medium. (D) Quantitative spectrophotometric biofilm assay following crystal violet staining in MSgg medium. Analysis of variance detected a significant main group effect between the wild type and clp mutants (b, P

    Article Snippet: To quantify biofilm growth, we applied the crystal violet staining method in 96-well polystyrene plates (Thermo) [ – ].

    Techniques: Incubation, Biofilm Production Assay, Staining

    Nonsecosteroidal VDR ligands are potent agonists in keratinocytes and PBMCs. ( A ) LY2108491 and LY2109866 are potent inhibitors of keratinocyte proliferation. KerTr cells plated in 96-well plates were dosed with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 72 hours at 37°C before BrdU incorporation into DNA was analyzed as a measure of cell proliferation. Results (mean α SEM) of experiments performed in triplicate are shown. ( B ) Nonsecosteroidal VDR ligands are potent inducers of CYP24 gene expression in keratinocytes. TaqMan quantitative RT-PCR (Q-PCR) was performed on total RNA prepared from KerTr cells treated with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. Levels of GAPDH mRNA were measured in all the samples, and the results were normalized and presented as fold induction (α SEM) compared with normalized CYP24 levels in vehicle-treated cells. ( C ) Nonsecosteroidal VDR ligands are efficacious in TPA- and PHA-activated PBMCs. Primary cells isolated from donors were stimulated with TPA (100 ng/ml) and PHA (25 μl/ml) and treated with vehicle or 100 nM each of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. TaqMan Q-PCR was performed on RNA obtained from vehicle-treated or VDR ligand–treated samples, using primer pairs and probes for IL-2, IL-4, IL-10, GATA3, and GAPDH. The amount of IL-2, IL-4, IL-10, and GATA3 transcripts relative to GAPDH transcripts is shown as mean α SEM of quadruplicate experimes.

    Journal: Journal of Clinical Investigation

    Article Title: Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators

    doi: 10.1172/JCI25901

    Figure Lengend Snippet: Nonsecosteroidal VDR ligands are potent agonists in keratinocytes and PBMCs. ( A ) LY2108491 and LY2109866 are potent inhibitors of keratinocyte proliferation. KerTr cells plated in 96-well plates were dosed with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 72 hours at 37°C before BrdU incorporation into DNA was analyzed as a measure of cell proliferation. Results (mean α SEM) of experiments performed in triplicate are shown. ( B ) Nonsecosteroidal VDR ligands are potent inducers of CYP24 gene expression in keratinocytes. TaqMan quantitative RT-PCR (Q-PCR) was performed on total RNA prepared from KerTr cells treated with various concentrations of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. Levels of GAPDH mRNA were measured in all the samples, and the results were normalized and presented as fold induction (α SEM) compared with normalized CYP24 levels in vehicle-treated cells. ( C ) Nonsecosteroidal VDR ligands are efficacious in TPA- and PHA-activated PBMCs. Primary cells isolated from donors were stimulated with TPA (100 ng/ml) and PHA (25 μl/ml) and treated with vehicle or 100 nM each of 1,25-(OH) 2 D 3 , LY2108491, or LY2109866 for 24 hours. TaqMan Q-PCR was performed on RNA obtained from vehicle-treated or VDR ligand–treated samples, using primer pairs and probes for IL-2, IL-4, IL-10, GATA3, and GAPDH. The amount of IL-2, IL-4, IL-10, and GATA3 transcripts relative to GAPDH transcripts is shown as mean α SEM of quadruplicate experimes.

    Article Snippet: Pooled splenocytes of 5 individual mice from the same group were plated in triplicate in a 96-well round-bottom plate at 2 × 105 cells per well in 200 μl complete RPMI 1640 medium (Invitrogen Corp.) supplemented with 2 mM l-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, 5.5 × 10–5 M 2-mercaptoethanol, and 5% FCS (all supplements from Invitrogen Corp.) containing 10 μg/ml MOG35–55 (Peptides International Inc.) or medium, and cultured at 37°C, 5% CO2 for 72 hours.

    Techniques: BrdU Incorporation Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Isolation

    Binding of wild-type α1 I domain to Toolkits II and III and to selected shorter triple-helical peptides. The recombinant wild-type α1 I domain was incubated in wells of Immulon 2HB 96-well plates coated with peptides, and adhesion was measured in the presence of either 2 m m Mg 2+ ( black bars ) or 2 m m EDTA ( white bars ) as described under “Experimental Procedures.” BSA, GPP10, and GFOGER served as control surface coatings. Experiments were performed in triplicate using 0.1 μg of protein/well. a , adhesion to Toolkit II; b , adhesion to Toolkit III; c , adhesion to selected shorter peptides, formatted with the indicated sequence flanked by (GPP) 5 host peptides. Long GFPGER has the same sequence as II-28 but with “O” in its GFOGER motif replaced with “P.” Data are the mean ± S.E. of at least six independent experiments. Col , collagen.

    Journal: The Journal of Biological Chemistry

    Article Title: Mapping of Potent and Specific Binding Motifs, GLOGEN and GVOGEA, for Integrin ?1?1 Using Collagen Toolkits II and III *

    doi: 10.1074/jbc.M112.353144

    Figure Lengend Snippet: Binding of wild-type α1 I domain to Toolkits II and III and to selected shorter triple-helical peptides. The recombinant wild-type α1 I domain was incubated in wells of Immulon 2HB 96-well plates coated with peptides, and adhesion was measured in the presence of either 2 m m Mg 2+ ( black bars ) or 2 m m EDTA ( white bars ) as described under “Experimental Procedures.” BSA, GPP10, and GFOGER served as control surface coatings. Experiments were performed in triplicate using 0.1 μg of protein/well. a , adhesion to Toolkit II; b , adhesion to Toolkit III; c , adhesion to selected shorter peptides, formatted with the indicated sequence flanked by (GPP) 5 host peptides. Long GFPGER has the same sequence as II-28 but with “O” in its GFOGER motif replaced with “P.” Data are the mean ± S.E. of at least six independent experiments. Col , collagen.

    Article Snippet: Peptides were coated at 10 μg/ml for 1 h at 22 °C on Immulon 2HB 96-well plates (Thermo Scientific) and blocked for 1 h with 200 μl of TBS containing 50 mg/ml BSA.

    Techniques: Binding Assay, Recombinant, Incubation, Sequencing

    Binding of PC12 cells to Toolkits II and III and selected peptides. Immulon 2HB 96-well plates or E-Plates TM for the xCELLigence instrument were coated with the indicated peptides as described under “Experimental Procedures.” a and b , PC12 cells were seeded at 10 5 /well, and SPBA was performed after 1 h as described using the colorimetric lactate dehydrogenase assay kit. c and d , PC12 cells were seeded onto E-Plates at 2.5 × 10 4 cells/well, and the cell index was measured continually. PC12 cell binding to Toolkits II ( a ) and III ( b ) is shown. PC12 cell adhesion to the indicated Toolkit peptides ( c ) and to selected shorter integrin-binding peptides ( d ) is shown as end point data after 2 h of adhesion. All experiments were performed in triplicate in the presence of either 2 m m Mg 2+ or EDTA as indicated. Data are the mean ± S.E. of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Mapping of Potent and Specific Binding Motifs, GLOGEN and GVOGEA, for Integrin ?1?1 Using Collagen Toolkits II and III *

    doi: 10.1074/jbc.M112.353144

    Figure Lengend Snippet: Binding of PC12 cells to Toolkits II and III and selected peptides. Immulon 2HB 96-well plates or E-Plates TM for the xCELLigence instrument were coated with the indicated peptides as described under “Experimental Procedures.” a and b , PC12 cells were seeded at 10 5 /well, and SPBA was performed after 1 h as described using the colorimetric lactate dehydrogenase assay kit. c and d , PC12 cells were seeded onto E-Plates at 2.5 × 10 4 cells/well, and the cell index was measured continually. PC12 cell binding to Toolkits II ( a ) and III ( b ) is shown. PC12 cell adhesion to the indicated Toolkit peptides ( c ) and to selected shorter integrin-binding peptides ( d ) is shown as end point data after 2 h of adhesion. All experiments were performed in triplicate in the presence of either 2 m m Mg 2+ or EDTA as indicated. Data are the mean ± S.E. of three independent experiments.

    Article Snippet: Peptides were coated at 10 μg/ml for 1 h at 22 °C on Immulon 2HB 96-well plates (Thermo Scientific) and blocked for 1 h with 200 μl of TBS containing 50 mg/ml BSA.

    Techniques: Binding Assay, Lactate Dehydrogenase Assay

    Crystal structures of LEDGF/IBD (A), BI-1001 (B), KF115 (C), and KF116 (D) bound to HIV-1 IN CCD. The IN subunit 1 and 2 are colored in cyan and green, respectively. LEDGF/IBD loop (amino acids 365–368) is shown in dark blue. BI-1001 is shown in orange. KF115 is shown in red. KF116 is shown in magenta. The hydrogen bonds between the IN subunit and the LEDGF/IBD or the indicated inhibitors are shown by black dashed lines. Side chains of HIV-1 IN residues A128T and T125 in subunit 1, and E170, H171 and T174 in subunit 2 are shown.

    Journal: PLoS Pathogens

    Article Title: A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

    doi: 10.1371/journal.ppat.1004171

    Figure Lengend Snippet: Crystal structures of LEDGF/IBD (A), BI-1001 (B), KF115 (C), and KF116 (D) bound to HIV-1 IN CCD. The IN subunit 1 and 2 are colored in cyan and green, respectively. LEDGF/IBD loop (amino acids 365–368) is shown in dark blue. BI-1001 is shown in orange. KF115 is shown in red. KF116 is shown in magenta. The hydrogen bonds between the IN subunit and the LEDGF/IBD or the indicated inhibitors are shown by black dashed lines. Side chains of HIV-1 IN residues A128T and T125 in subunit 1, and E170, H171 and T174 in subunit 2 are shown.

    Article Snippet: Briefly, 107 copies of total viral RNA were incubated with 50 ng of purified HIV-1 RT (NIH Catalog # 3555) at 37°C for 15 min in 20 µl of RT buffer containing 50 mM Tris-HCl (pH 7.5), 60 mM KCl, 3 mM MgCl2 , 5 mM DTT, 10 U of RNaseOut (Invitrogen), 200 µM dCTP, 200 µM dTTP, 50 µM ddATP and 5 µCi of [α-32 P] dGTP.

    Techniques:

    KF116 promotes IN multimerization in vitro and in HIV-1 particles. ( A ) DLS analysis of KF116-induced multimerization of recombinant IN. Size distribution of IN at 2 minutes (blue), 8 minutes (red), 30 minutes (green) and 120 minutes (yellow) after addition of KF116. No detectable signal was recorded in control experiments with KF116 alone or IN+DMSO incubated for up to 120 minutes. ( B ) The schematic to show the inhibitor induced equilibrium shift toward aberrant IN oligomerization. ( C ) HIV-1 virions were produced in HEK293T cells in the presence of DMSO or 1 µM KF116, cell-free virions were harvested, detergent-lysed, and treated with BS 3 cross-linking reagent. The indicated volumes of cross-linked reaction products were resolved by SDS-PAGE and immunoblotted with HIV-1 IN antibody. The bands corresponding to IN “monomer”, “dimer”, and “higher order oligomers” are indicated.

    Journal: PLoS Pathogens

    Article Title: A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

    doi: 10.1371/journal.ppat.1004171

    Figure Lengend Snippet: KF116 promotes IN multimerization in vitro and in HIV-1 particles. ( A ) DLS analysis of KF116-induced multimerization of recombinant IN. Size distribution of IN at 2 minutes (blue), 8 minutes (red), 30 minutes (green) and 120 minutes (yellow) after addition of KF116. No detectable signal was recorded in control experiments with KF116 alone or IN+DMSO incubated for up to 120 minutes. ( B ) The schematic to show the inhibitor induced equilibrium shift toward aberrant IN oligomerization. ( C ) HIV-1 virions were produced in HEK293T cells in the presence of DMSO or 1 µM KF116, cell-free virions were harvested, detergent-lysed, and treated with BS 3 cross-linking reagent. The indicated volumes of cross-linked reaction products were resolved by SDS-PAGE and immunoblotted with HIV-1 IN antibody. The bands corresponding to IN “monomer”, “dimer”, and “higher order oligomers” are indicated.

    Article Snippet: Briefly, 107 copies of total viral RNA were incubated with 50 ng of purified HIV-1 RT (NIH Catalog # 3555) at 37°C for 15 min in 20 µl of RT buffer containing 50 mM Tris-HCl (pH 7.5), 60 mM KCl, 3 mM MgCl2 , 5 mM DTT, 10 U of RNaseOut (Invitrogen), 200 µM dCTP, 200 µM dTTP, 50 µM ddATP and 5 µCi of [α-32 P] dGTP.

    Techniques: In Vitro, Recombinant, Incubation, Produced, SDS Page

    LEDGF/p75 expression does not affect KF116 potency during late stage of HIV-1 replication. ( A ) Equivalent whole cell lysates from the clonal TALEN-derived PSIP 1 KO cell line (indicated as “KO”) and parental wild type HEK293T cell line (indicated as “WT”) were subjected to SDS-PAGE and immunoblotted for LEDGF/p75 and a GAPDH control to verify knockdown of LEDGF/p75 protein. ( B ) Dose-response curves representing the antiviral assays performed in WT or KO cell lines under the indicated conditions of drug treatment. For producer cell treatment, the VSV-G pseudotyped HIV-1-Luc progeny virions were prepared in the indicated cell line in the presence of KF116 and were then used to infect untreated HEK293T cells. For target cell treatment, KF116 was added directly to the indicated cell line and the cells were infected with untreated VSV-G pseudotyped HIV-1-Luc virions. ( C ) EC 50 values for the indicated antiviral assays. Results represent mean ± SD from three independent experiments.

    Journal: PLoS Pathogens

    Article Title: A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

    doi: 10.1371/journal.ppat.1004171

    Figure Lengend Snippet: LEDGF/p75 expression does not affect KF116 potency during late stage of HIV-1 replication. ( A ) Equivalent whole cell lysates from the clonal TALEN-derived PSIP 1 KO cell line (indicated as “KO”) and parental wild type HEK293T cell line (indicated as “WT”) were subjected to SDS-PAGE and immunoblotted for LEDGF/p75 and a GAPDH control to verify knockdown of LEDGF/p75 protein. ( B ) Dose-response curves representing the antiviral assays performed in WT or KO cell lines under the indicated conditions of drug treatment. For producer cell treatment, the VSV-G pseudotyped HIV-1-Luc progeny virions were prepared in the indicated cell line in the presence of KF116 and were then used to infect untreated HEK293T cells. For target cell treatment, KF116 was added directly to the indicated cell line and the cells were infected with untreated VSV-G pseudotyped HIV-1-Luc virions. ( C ) EC 50 values for the indicated antiviral assays. Results represent mean ± SD from three independent experiments.

    Article Snippet: Briefly, 107 copies of total viral RNA were incubated with 50 ng of purified HIV-1 RT (NIH Catalog # 3555) at 37°C for 15 min in 20 µl of RT buffer containing 50 mM Tris-HCl (pH 7.5), 60 mM KCl, 3 mM MgCl2 , 5 mM DTT, 10 U of RNaseOut (Invitrogen), 200 µM dCTP, 200 µM dTTP, 50 µM ddATP and 5 µCi of [α-32 P] dGTP.

    Techniques: Expressing, Derivative Assay, SDS Page, Infection

    Virions produced in the presence of KF116 are defective in reverse transcription. ( A–E ) VSV-G pseudotyped HIV-1-Luc produced in the presence of DMSO, 1 µM KF116, or 1 µM RAL were used to infect HEK293T cells. Infected cells were harvested at the indicated times and subjected to quantitative PCR (qPCR) or luciferase assay. Graphs indicate the amount of PCR products relative to non-treated (DMSO) sample at 6 h post-infection for ( A ) early reverse transcription (Early RT), ( B ) late reverse transcription (Late RT) and ( C ) 2-LTR circles (2-LTRs) products. ( D ) Bar graphs indicate the integrated provirus relative to non-treated (DMSO) control at 7 days post-infection. ( E ) Aliquots of infected cells were harvested and luciferase assay was performed at 48 h post-infection. The luciferase signal obtained for the non-treated (DMSO) sample was set to 100%. All graphs represent mean ± SD ( n = 3). ( F–J ) HEK293T cells were treated with DMSO, 1 µM KF116, or 1 µM RAL and then infected with VSV-G pseudotyped HIV-1-Luc. Infected cells were harvested at the indicated times and subjected to qPCR or luciferase assay. Graphs indicate the amount of PCR products relative to non-treated (DMSO) sample at 6 h post-infection for ( F ) early reverse transcription (Early RT), ( G ) late reverse transcription (Late RT) and ( H ) 2-LTR circles (2-LTRs) products. ( I ) Bar graphs indicate the integrated provirus relative to non-treated (DMSO) sample at 7 days post-infection. ( J ) Aliquots of infected cells were harvested and luciferase assay was performed at 48 h post-infection. The luciferase signal obtained for the non-treated (DMSO) sample was set to 100%. All graphs represent mean ± SD ( n = 3).

    Journal: PLoS Pathogens

    Article Title: A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

    doi: 10.1371/journal.ppat.1004171

    Figure Lengend Snippet: Virions produced in the presence of KF116 are defective in reverse transcription. ( A–E ) VSV-G pseudotyped HIV-1-Luc produced in the presence of DMSO, 1 µM KF116, or 1 µM RAL were used to infect HEK293T cells. Infected cells were harvested at the indicated times and subjected to quantitative PCR (qPCR) or luciferase assay. Graphs indicate the amount of PCR products relative to non-treated (DMSO) sample at 6 h post-infection for ( A ) early reverse transcription (Early RT), ( B ) late reverse transcription (Late RT) and ( C ) 2-LTR circles (2-LTRs) products. ( D ) Bar graphs indicate the integrated provirus relative to non-treated (DMSO) control at 7 days post-infection. ( E ) Aliquots of infected cells were harvested and luciferase assay was performed at 48 h post-infection. The luciferase signal obtained for the non-treated (DMSO) sample was set to 100%. All graphs represent mean ± SD ( n = 3). ( F–J ) HEK293T cells were treated with DMSO, 1 µM KF116, or 1 µM RAL and then infected with VSV-G pseudotyped HIV-1-Luc. Infected cells were harvested at the indicated times and subjected to qPCR or luciferase assay. Graphs indicate the amount of PCR products relative to non-treated (DMSO) sample at 6 h post-infection for ( F ) early reverse transcription (Early RT), ( G ) late reverse transcription (Late RT) and ( H ) 2-LTR circles (2-LTRs) products. ( I ) Bar graphs indicate the integrated provirus relative to non-treated (DMSO) sample at 7 days post-infection. ( J ) Aliquots of infected cells were harvested and luciferase assay was performed at 48 h post-infection. The luciferase signal obtained for the non-treated (DMSO) sample was set to 100%. All graphs represent mean ± SD ( n = 3).

    Article Snippet: Briefly, 107 copies of total viral RNA were incubated with 50 ng of purified HIV-1 RT (NIH Catalog # 3555) at 37°C for 15 min in 20 µl of RT buffer containing 50 mM Tris-HCl (pH 7.5), 60 mM KCl, 3 mM MgCl2 , 5 mM DTT, 10 U of RNaseOut (Invitrogen), 200 µM dCTP, 200 µM dTTP, 50 µM ddATP and 5 µCi of [α-32 P] dGTP.

    Techniques: Produced, Infection, Real-time Polymerase Chain Reaction, Luciferase, Polymerase Chain Reaction

    KF116 selectively impairs the late stage of HIV-1 replication. ( A ) Dose-response curves for KF116 antiviral activities during early stage, late stage or one full replication cycle. For early stage experiments, KF116 was added directly to the target cells and then these cells were infected with untreated virions. For late stage experiments, the progeny virions were prepared in the presence of KF116 and were then used to infect untreated target cells. For one full replication cycle experiments, KF116 was added to both producer and target cells. ( B ) EC 50 values for the indicated antiviral assays. Results represent mean ± SD from three independent experiments.

    Journal: PLoS Pathogens

    Article Title: A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

    doi: 10.1371/journal.ppat.1004171

    Figure Lengend Snippet: KF116 selectively impairs the late stage of HIV-1 replication. ( A ) Dose-response curves for KF116 antiviral activities during early stage, late stage or one full replication cycle. For early stage experiments, KF116 was added directly to the target cells and then these cells were infected with untreated virions. For late stage experiments, the progeny virions were prepared in the presence of KF116 and were then used to infect untreated target cells. For one full replication cycle experiments, KF116 was added to both producer and target cells. ( B ) EC 50 values for the indicated antiviral assays. Results represent mean ± SD from three independent experiments.

    Article Snippet: Briefly, 107 copies of total viral RNA were incubated with 50 ng of purified HIV-1 RT (NIH Catalog # 3555) at 37°C for 15 min in 20 µl of RT buffer containing 50 mM Tris-HCl (pH 7.5), 60 mM KCl, 3 mM MgCl2 , 5 mM DTT, 10 U of RNaseOut (Invitrogen), 200 µM dCTP, 200 µM dTTP, 50 µM ddATP and 5 µCi of [α-32 P] dGTP.

    Techniques: Infection

    Genotype of HIV-1 variants selected in cell culture in the presence of KF116. ( A ) Mutations in the HIV-1 NL4-3 IN gene of resistant viruses selected with KF116. Clonal sequencing of viral passage was carried out at passages 5 and 10, respectively. Eighty-two clones from each viral passage were sequenced using three sequencing primers covering the entire IN gene. Percentage of IN mutations for a given passage are indicated. Passage 5 corresponds to 50 days of selection with the KF116 concentration reaching 0.8 µM. Passage 10 corresponds to 100 days of selection with the KF116 concentration reaching 25.6 µM. ( B ) Crystal structure of KF116 bound to HIV-1 IN CCD dimer indicating the Thr-124, Val-165 and Thr-174 residues. The IN subunit 1 and 2 are colored in cyan and green, respectively. KF116 is shown in magenta.

    Journal: PLoS Pathogens

    Article Title: A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

    doi: 10.1371/journal.ppat.1004171

    Figure Lengend Snippet: Genotype of HIV-1 variants selected in cell culture in the presence of KF116. ( A ) Mutations in the HIV-1 NL4-3 IN gene of resistant viruses selected with KF116. Clonal sequencing of viral passage was carried out at passages 5 and 10, respectively. Eighty-two clones from each viral passage were sequenced using three sequencing primers covering the entire IN gene. Percentage of IN mutations for a given passage are indicated. Passage 5 corresponds to 50 days of selection with the KF116 concentration reaching 0.8 µM. Passage 10 corresponds to 100 days of selection with the KF116 concentration reaching 25.6 µM. ( B ) Crystal structure of KF116 bound to HIV-1 IN CCD dimer indicating the Thr-124, Val-165 and Thr-174 residues. The IN subunit 1 and 2 are colored in cyan and green, respectively. KF116 is shown in magenta.

    Article Snippet: Briefly, 107 copies of total viral RNA were incubated with 50 ng of purified HIV-1 RT (NIH Catalog # 3555) at 37°C for 15 min in 20 µl of RT buffer containing 50 mM Tris-HCl (pH 7.5), 60 mM KCl, 3 mM MgCl2 , 5 mM DTT, 10 U of RNaseOut (Invitrogen), 200 µM dCTP, 200 µM dTTP, 50 µM ddATP and 5 µCi of [α-32 P] dGTP.

    Techniques: Cell Culture, Sequencing, Clone Assay, Selection, Concentration Assay

    KF116 impairs formation of dense cores in HIV-1 virions. ( A ) Representative thin-section electron micrographs of HIV-1 virions produced in the presence of DMSO or 1 µM KF116. ( B ) Quantitative analysis of mature virions prepared in the presence of DMSO or 1 µM KF116. Correctly matured electron dense cores are shown in black and eccentric virions lacking electron density are shown in gray. Standard errors determined from two independent experiments are shown. Images of at least 50 mature virions were examined from each experiment. ( C ) Sucrose density gradient fractionation of detergent-lysed HIV-1 virions produced in HEK293T cells in the presence of DMSO or 1 µM KF116. Cell-free virions were harvested, detergent-lysed, and separated on 30–70% linear sucrose density gradients. Twenty-one 0.5 ml fractions were collected from the top of the gradient and subjected to SDS-PAGE and immunoblotted with HIV-1 Gag antisera to monitor the distribution of HIV-1 capsid. Positions of Gag p24 (capsid) and Gag p17 (matrix) are indicated. ( D ) Quantitation of HIV-1 capsid (p24) signal intensity from (B) as measured by ImageJ software. Graph represents the relative distribution of HIV-1 capsid (p24) in each of the sucrose density gradient fractions.

    Journal: PLoS Pathogens

    Article Title: A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

    doi: 10.1371/journal.ppat.1004171

    Figure Lengend Snippet: KF116 impairs formation of dense cores in HIV-1 virions. ( A ) Representative thin-section electron micrographs of HIV-1 virions produced in the presence of DMSO or 1 µM KF116. ( B ) Quantitative analysis of mature virions prepared in the presence of DMSO or 1 µM KF116. Correctly matured electron dense cores are shown in black and eccentric virions lacking electron density are shown in gray. Standard errors determined from two independent experiments are shown. Images of at least 50 mature virions were examined from each experiment. ( C ) Sucrose density gradient fractionation of detergent-lysed HIV-1 virions produced in HEK293T cells in the presence of DMSO or 1 µM KF116. Cell-free virions were harvested, detergent-lysed, and separated on 30–70% linear sucrose density gradients. Twenty-one 0.5 ml fractions were collected from the top of the gradient and subjected to SDS-PAGE and immunoblotted with HIV-1 Gag antisera to monitor the distribution of HIV-1 capsid. Positions of Gag p24 (capsid) and Gag p17 (matrix) are indicated. ( D ) Quantitation of HIV-1 capsid (p24) signal intensity from (B) as measured by ImageJ software. Graph represents the relative distribution of HIV-1 capsid (p24) in each of the sucrose density gradient fractions.

    Article Snippet: Briefly, 107 copies of total viral RNA were incubated with 50 ng of purified HIV-1 RT (NIH Catalog # 3555) at 37°C for 15 min in 20 µl of RT buffer containing 50 mM Tris-HCl (pH 7.5), 60 mM KCl, 3 mM MgCl2 , 5 mM DTT, 10 U of RNaseOut (Invitrogen), 200 µM dCTP, 200 µM dTTP, 50 µM ddATP and 5 µCi of [α-32 P] dGTP.

    Techniques: Produced, Fractionation, SDS Page, Quantitation Assay, Software

    Effects of MINI KF116 and ALLINI GS-B on LEDGF/p75-dependent targeting of HIV-1 integration site distribution on chromatin. ( A ) HIV-1 integration frequencies in RefSeq and known genes, comparing effects of KF116 and GS-B on LEDGF/p75-dependent targeting of HIV-1 integration. All the samples differed significantly from their respective matched random controls (using Fisher's exact test, P

    Journal: PLoS Pathogens

    Article Title: A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

    doi: 10.1371/journal.ppat.1004171

    Figure Lengend Snippet: Effects of MINI KF116 and ALLINI GS-B on LEDGF/p75-dependent targeting of HIV-1 integration site distribution on chromatin. ( A ) HIV-1 integration frequencies in RefSeq and known genes, comparing effects of KF116 and GS-B on LEDGF/p75-dependent targeting of HIV-1 integration. All the samples differed significantly from their respective matched random controls (using Fisher's exact test, P

    Article Snippet: Briefly, 107 copies of total viral RNA were incubated with 50 ng of purified HIV-1 RT (NIH Catalog # 3555) at 37°C for 15 min in 20 µl of RT buffer containing 50 mM Tris-HCl (pH 7.5), 60 mM KCl, 3 mM MgCl2 , 5 mM DTT, 10 U of RNaseOut (Invitrogen), 200 µM dCTP, 200 µM dTTP, 50 µM ddATP and 5 µCi of [α-32 P] dGTP.

    Techniques: