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| 1. |
Emonet, S.F., Garidou, L., McGavern, D.B. and de la Torre, J.C.
(2009)
Generation of recombinant lymphocytic chriomeningits virus with trisegmented genomes stably expressing two additional genes of interest.
Proc. Natl. Acad. Sci. USA
106
,
3473-3478
.
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Notes:
The lymphocytic choriomeningitis virus (LCMV) was used as a model to create a trisegmented recombinant arenavirus in which viral genes were replaced by a gene of interest. One such engineered virus, r3LCMV CAT/FLuc, was used in a pilot screen to identify anti-arenaviral compounds. Firefly luciferase (FLuc) activity was measured using the ONE-Glo™ Luciferase Assay System.
(0003957) |
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Products: ONE-Glo™ Luciferase Assay System |
| 2. |
Xia, M., Huang, R., Guo, V., Southall, N., Cho, M.H., Inglese, J., Austin, C.P. and Nirenberg, M.
(2009)
Identification of compounds that potentiate CREB signaling as possible enhancers of long-term memory.
Proc. Natl. Acad. Sci. U S A
106
,
2412–7
.
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Notes:
In this study, small molecule enhancers of cAMP response element binding (CREB) were studied using quantitative high-throughput screening. After an initial screen of 73,000 compounds, 1,800 compounds were classified as potentiators of CREB activity. A second screening to confirm the compound potential was performed using the GloResponse™ CRE-luc2P HEK293 Cell Line. Five microliters of cells in assay medium were seeded in 1,536-well plates at a density of 2,500 cells/well. The next day, 23 nl of compound in DMSO or DMSO alone was dispensed into each well, then 1 μl of NKH477 (final concentration, 200 nM) or media alone was added to the assay plates. After incubating the cells for 4 hours at 37 °C, 6 μl of Bright-Glo™ Luciferase Assay Reagent was added to each well, incubated at room temperature for 10 minutes and the luminescence measured.
(0004004) |
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Products: Bright-Glo™ Luciferase Assay System | GloResponse™ CRE-luc2P HEK293 Cell Line |
| 3. |
Hawes, J.J., Nerva, J.D. and Reily, K.M.
(2008)
Novel dual-reporter preclinical screen for antiastrocytoma agents identifies cytostatic and cytotoxic compounds
J. Biolmol. Scr.
13
,
795-803
.
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Notes:
The authors of this paper created a mouse model for in vitro assays to screen for therapeutic compounds specifically active against astrocytic gliomas. A Green/Red luciferase (G/R-luc) dual-reporter system was created in KR158 cells (derived from grade III agressive mouse anaplastic astrocytoma). The green click beetle luciferase gene (from pCBC68-Basic Vector) was placed under the control of the E2F1 promoter, and the red click beetle luciferase gene (from pCBR-Basic Vector) was placed under the control of the CMV promoter. The dual-reporter assay simultaneously evaluates E2F1 promoter activity and assesses cytotocity; the assay also distinguishes cytostatic from cytotoxic compounds.
(0003949) |
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Products: Chroma-Glo™ Luciferase Assay System | pCBG68-Basic Vector | pCBR-Basic Vector |
| 4. |
Liu, Q., Fu, H., Sun, F., Zhang, H., Tie, Y., Zhu, J., Xing, R., Sun, Z. and Zheng, X.
(2008)
miR-16 family induces cell cycle arrest by regulating multiple cell cycle genes.
Nucleic Acids Res.
36
,
5391–404
.
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Notes:
To identify microRNA targets, the authors created a Drosha-knockdown cell line and confirmed depletion of Drosha and three randomly selected miRNAs in these cells by quantitative RT-PCR, using β-actin as a control. The reverse transcription step was performed using the ImProm-II™ Reverse Transcription System. The authors then performed microarray analysis to monitor expression of transcripts to determine which were upregulated as a result of Drosha depletion; cRNA used in these microarray experiments was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. Cyclin D1 was identified as a potential miRNA target. To screen miRNAs that regulate cyclin D1, the authors cloned the cyclin D1 3´ untranslated region downstream of the firefly luciferase gene of the pGL3-Control Vector and measured luciferase levels in transfected cells using the Dual Luciferase Reporter Assay System. Renilla luciferase in the pRL-TK Vector was used as a normalization control.
(0003894) |
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Products: Dual-Luciferase® Reporter Assay System | ImProm-II™ Reverse Transcriptase | pGL3-Control Vector | pRL-TK Vector | T7 RiboMAX™ Express Large Scale RNA Production System |
| 5. |
Yoon, J.-J., Chawla, D., Pall, T., Ndungu, M., Du, Y., Kurtkaya, S., Sun, A., Snyder, J.P. and Plemper, R.K.
(2008)
High-throughput screening-based identification of paramyxovirus inhibitors.
J. Biomol. Screen
13
,
591-608
.
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Notes:
The authors describe an HTS assay to screen for inhibitors of measles virus infection. The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess toxicity of all confirmed hit compounds from the primary screen. A luminescent assay was used to assess cell-to-cell fusion in the presence candidate compounds that appeared to inhibit entry. Effector cells that expressed the T7 polymerase and measles virus H and F envelope proteins were overlaid on target cells expressing firefly luciferase under the control of a T7 polymerase promoter. Inhibition of fusion should reduce luciferase expression compared to a positive fusion control. Ten of the eleven compounds tested caused a dose-dependent reduction in luciferase expression, suggesting they block viral entry into cells. Luminescence was detected using the Bright-Glo™ Assay System.
(0003933) |
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Products: Bright-Glo™ Luciferase Assay System | CytoTox 96® Non-Radioactive Cytotoxicity Assay |
| 6. |
Betzi S, Restouin A, Opi S, Arold ST, Parrot I, Guerlesquin F, Morelli X, Collette Y.
(2007)
Protein protein interaction inhibition (2P2I) combining high throughput and virtual screening: Application to the HIV-1 Nef protein.
Proc. Natl. Acad. Sci. U S A
104
,
19256-19261
.
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Notes:
The authors wanted to screen inhibitory compounds for the HIV-1 accessory protein Nef using both computer modeling and experimental methods. Using a structure-based program for the SH3 binding surface of Nef, drug compounds were screened in silico and then further analyzed using a cell-based assay. The Nef gene and SH3 domain were cloned into the pACT and pBIND Vectors of the CheckMate™ Mammalian Two-Hybrid System, transfected into COS-7 cells, and 18 hours later, the cells exposed to potential inhibitors. After 24 hours, luciferase activity was assessed using either the Dual-Glo™ or the Steady-Glo® Luciferase Assay Systems.
(0003751) |
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Products: CheckMate™ Mammalian Two-Hybrid System | Dual-Glo® Luciferase Assay System | Steady-Glo® Luciferase Assay System |
| 7. |
Cho, Y.Y., Yao, K., Bode, A.M., Bergen, H.R. 3rd, Madden, B.J., Oh, S.M., Ermakova, S., Kang, B.S., Choi, H.S., Shim, J.H. and Dong, Z.
(2007)
RSK2 Mediates Muscle Cell Differentiation through Regulation of NFAT3.
J. Biol. Chem.
282
,
8380–8392
.
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Notes:
The CheckMate™ Mammalian Two-Hybrid System was used to screen for protein-binding partners for RSK2, a ribosomal S6 kinase involved in myoblast differentiation. The RSK2 cDNA was cloned into the pBIND Vector as bait while several transcription factors were amplified by PCR and cloned into the pACT Vector. The bait pBIND-RSK2 construct, the pACT-transcription factors and pG5-luc Vector were transfected into 293 cells at a 1:1:1 molar ratio. To assess the protein interaction, the cells were lysed, and the firefly luciferase activity was normalized to Renilla luciferase activity. The strongest interaction was with NFAT3.
(0003574) |
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Products: CheckMate™ Mammalian Two-Hybrid System |
| 8. |
Borawski, J., Lindeman, A., Buxton, F., Labow, M. and Gaither, L.A.
(2007)
Optimization procedure for small interfering RNA transfection in a 384-well format.
J. Biomol. Screen.
12
,
546–559
.
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Notes:
A lentiviral expression vector containing the firefly luciferase gene from a pGL3 Vector was transduced into SKOV3 cells in 384-well plates, transfected with various siRNAs and analyzed 72 hours later. The luciferase expression was determined using the Bright-Glo™ Luciferase Assay System and cell viability assessed using the CellTiter-Glo® Luminescent Cell Viability Assay.
(0003729) |
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Products: Bright-Glo™ Luciferase Assay System | CellTiter-Glo® Luminescent Cell Viability Assay |
| 9. |
Galkin, A.V., Melnick, J.S., Kim, S., Hood, T.L., Li, N., Li, L., Xia, G., Steensma, R., Chopiuk, G., Jiang, J., Wan, Y., Ding, P., Liu, Y., Sun, F., Schultz, P.G., Gray, N.S. and Warmuth, M.
(2007)
Identification of NVP-TAE684, a potent, selective and efficacious inhibitor of NPM-ALK
Proc. Natl. Acad. Sci. USA
104
,
270-275
.
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Notes:
NVP-TAE684 was identified as an inhibitor of the constitutive anaplastic lymphoma kinase (ALK) activity associated with the NPM-ALK fusion. NPM-ALK fusion protein is created by translocation event characteristic of anaplastic large-cell lymophomas. NVP-TAE684 was screened against a panel of 35 Ba/F3 cell lines expressing a variety of tyrosine kinases constitutively activated by fusion to TEL. The CellTiter-Glo® Luminescent Cell Viability Assay was used to detect any decrease in viability as a result of treatment with NVP-TAE684. The inhibitory effect of NVP-TAE684 was specific for ALK-associated cell proliferation. In a secondary screen to determine potency, TAE684 was screened against two human anaplastic large-cell lymphoma cell lines. Inhibition of proliferation correlated with dosage and was assessed using the Bright-Glo® Luciferase Assay System.
(0003738) |
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Products: Bright-Glo™ Luciferase Assay System | CellTiter-Glo® Luminescent Cell Viability Assay |
| 10. |
Ji, C., Zhang, J., Cammack, N. and Sankuratri, S.
(2006)
Development of a novel dual CCR5-dependent and CXCR4-dependent cell-cell fusion assay system with inducible gp160 expression.
J. Biomol. Screen.
11
,
65–74
.
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Notes:
The effector cells of a high-throughput cell-cell fusion assay were dispensed in 384-well plates and expression of the envelope gene induced. Small-molecule compounds or antibodies being tested for their ability to prevent cell fusion were added to the effector cells (carrying firefly luciferase under control of HIV-2 LTR) prior to adding the target cells. The cells were cocultured for 20–24 hours, and luciferase expression was measured using the Steady-Glo® Luciferase Assay System.
(0003731) |
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Products: Steady-Glo® Luciferase Assay System |
| 11. |
Radhakrishnan, S.K., Bhat, U.G., Hughes, D.E., Wang, I.C., Costa, R.H. and Gartel, A.L.
(2006)
Identification of a chemical inhibitor of the oncogenic transcription factor forkhead box M1.
Cancer Res
66
,
9731–9735
.
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Notes:
To screen a compound library for an inhibitor of FoxM1 transcriptional activity, U2OS cells with doxycyclin-inducible FoxM1-green fluorescent protein (GFP) fusion protein were stably transfected with a firefly luciferase construct driven by a 6× FoxM1 responsive promoter and the pRL-CMV Vector. These cells were grown overnight in 96-well plates, treated with doxycyclin and test compounds. Firefly and Renilla luciferase expression was assessed using the Dual-Glo™ Luciferase Assay System.
(0003730) |
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Products: Dual-Glo® Luciferase Assay System |
| 12. |
Bernstein, D., Hook, B., Hajarnavis, A., Opperman, L. and Wickens, M.
(2006)
Binding specificity and mRNA targets of a C. elegans PUF protein, FBF-1.
RNA
11
,
447–458
.
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Notes:
PUF proteins act to reduce expression upon binding to the 3´UTR of target mRNA sequences. In this study, the binding of various PUF proteins (including C. elegans FBF-1) to known PUF binding sites was evaluated using a yeast three-hybrid system. For this assay, various PUF proteins fused to the Gal4 activation domain were expressed in pACT and pACT2 plasmids, and DNA oligos expressing PUF binding RNA sequences were cloned into the plasmid pIIIA/MS2-2. Assays were then performed in the yeast strain YBZ-1. β-galactosidase expression, indicating interaction between each PUF protein and the various binding sites, was measured using both solid- (colony lift) and liquid-phase assays. For the liquid-phase assays, β-galactosidase expression was measured using the Beta-Glo® Assay System. Cells were grown in selective media to an O.D.600 of 0.1–0.3, then mixed with an equal volume of Beta-Glo® Assay Reagent. After one hour, luminescence was measured. Luminescence values were normalized to cell number. The results of these binding assays were used to formulate a consensus PUF binding sequence, which was then used to screen a C. elegans 3´ UTR database for potential FBF-1 target sequences.
(0003458) |
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Products: Beta-Glo® Assay System |
| 13. |
Katso, R.M., Parham, J.H., Caivono, M., Clay, W.C., Condreay, J.P., Gray, D.W., Lindley, K.M., Mason, S.J., Rieger, J., Wakes, N.C., Cairns, W.J. and Merrihew, R.V.
(2005)
Evaluation of cell-based assays for steroid nuclear receptors delivered by recombinant baculoviruses.
J. Biomol. Screen.
10
,
715–724
.
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Notes:
The authors used modified baculoviruses containing mammalian expression cassettes in cell-based reporter assays for nuclear receptor activity. An MMLV-firefly luciferase was used as a reporter construct. Activity of the reporter was assessed using the Steady-Glo® Reagent.
(0003945) |
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Products: Steady-Glo® 1X Solution |
| 14. |
Bakshi, P., Liao, Y-F., Gao, J., Ni, J., Stein, R., Yeh, L-A., Wolfe, M.S.
(2005)
A high-throughput screen to identifiy inhibitors of amyloid beta-protein precursor processing
J. Biomol. Screen.
10
,
1-12
.
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Notes:
A key component in the pathogenesis of Alzheimer’s disease is cerebral accumulation of amyloid-beta protein (Aβ). Aβ is produced by proteolysis of amyloid-β-protein precursor (APP) by ß- and gamma-secretases, thus these enzymes are considered important drug targets for Alzheimer’s disease. Existing assays for assessing inhibition of alpha-, beta- and gamma-secretases include HPLC or ELISA assays that are cumbersome, expensive and not well-suited to high-throughput screening. The authors developed a luciferase reporter system to identify new molecules that inhibit APP processing. They then successfully interfaced this sensitive, specific and quantitative assay with a high-throughput screen, useful for identifying both inhibitors and stimulators of APP processing.
(0003775) |
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Products: Bright-Glo™ Luciferase Assay System | CytoTox-ONE™ Homogeneous Membrane Integrity Assay | CytoTox-ONE™ Homogeneous Membrane Integrity Assay, HTP | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | Passive Lysis 5X Buffer | pRL-TK Vector |
| 15. |
Wang, Y., Klock,H., Yin, H., Wolff, K., Bieza, K., Niswonger, K., Matzen, J., Gunderson, D., Hale, J, Lesley, S., Kuhen, K., Caldwell, J, Brinker, A.
(2005)
Homogeneous high-throughput screening assays for HIV-1 integrase 3' processing and strand transfer activities
J. Biomol. Screen.
10
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456-462
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Notes:
HIV-1 integrase (HIV-IN) catalyzes a two-step reaction that results in HIV-1 provirus incorporation into the host cell genome. The steps involve an endonucleolytic 3’-processing (3P) followed by a strand transfer (ST) reaction. In past research it has been demonstrated that small molecule inhibitors of the in vitro activity of the 3P reaction only, lack antiviral activity, while inhibition of the HIV-IN ST reaction has been shown to be the key to effective suppression of viral replication in vivo. In the ST inhibitor realm, two series of diketo acids and naphthridine carboxamides have demonstrated antiviral activity in cell culture-based models. However, no compounds have completed clinical trials so far, due to either limited potency or high toxicity. Traditional integrase assays have been low-throughput, gel-based assays involving radiolabeled oligonucleotides. More recently high-throughput assays have been developed, but these microtiter-based assays, while amenable to automation, are complicated and labor intensive due to the need for plate coating and washing. The authors describe development of two robust, homogeneous time-resolved fluorescent energy transfer (TR-FRET)-based assays for HIV-IN 3P and ST reactions that are optimized for 384-well amd 1536-well plate formats. A screen for HIV-IN inhibitors was performed on a 1.36 x 106 compound library, resulting in a series of novel HIV-IN inhibitors that preferentially block integrase ST activity and show potential for further development as new antiviral drugs.
(0003776) |
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Products: Bright-Glo™ Luciferase Assay System |
| 16. |
Pratt, S.D., David, C.A., Black-Schaefer, C., Dandliker, P.J., Xuei, X., Warrior, U., Burns, D.J., Zhong, P., Cao, Z., Saiki, A.Y.C., Lerner, C.G., Chovan, L.E., Soni, N.B., Nilius, A.M., Wagenaar, F.L., Merta, P.J., Traphagen, L. and Beutel, B.A.
(2004)
A strategy for discovery of novel broad-spectrum antibacterials using a high-throughput Streptococcus pneumoniae transcription/translation screen.
J. Biomol. Screen.
9
,
3-11
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Notes:
Several different sequences from a S. pneumoniae pA promoter region were cloned into the pSP-luc+ vector and screened for expression levels in in vitro transcription/translation systems. The RiboMAX™ SP6 Large Scale RNA Production System was used to transcribe luciferase encoding mRNAs. Luciferase mRNAs and plasmids were used as templates in high throughput inhibition studies in an S. pneumoniae S30 extract described in the paper as well as in Promega’s E. Coli S30 Extract System for Circular DNA and in Rabbit Reticulocyte Lysate. Promega amino acid mixtures were also used in these studies.
(0003227) |
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Products: E. coli S30 Extract System for Circular DNA | Amino Acid Mixture, Complete | Rabbit Reticulocyte Lysate System, Nuclease Treated | RiboMAX™ Large Scale RNA Production System—SP6 |
| 17. |
Zheng Q., Yin G., Yan C., Cavet M. and Berk B.C.
(2004)
14-3-3beta binds to big mitogen-activated protein kinase 1 (BMK1/ERK5) and regulates BMK1 function.
J. Biol. Chem.
279(10)
,
8787-8791
.
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Notes:
The authors performed a yeast two-hybrid screen using big mitogen-activated kinase 1 (BMK1/ERK5) as the bait and identified the scaffolding protein 14-3-3beta. To confirm this interaction, the cloned mouse BMK1 gene was expressed in the TNT® T7 Quick Coupled Transcription/Translation System. The expressed protein was labeled with Transcend™ tRNA. Using a GST-14-3-3beta fusion protein, a pull-down assay was performed and the direct binding confirmed after immunoblotting and staining with streptavidin-horseradish peroxidase (HRP). The interaction of various BMK1 mutants were tested in a mammalian two-hybrid system and measured by the Dual-Luciferase® Reporter Assay System.
(0003078) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | TNT® T7 Quick Coupled Transcription/Translation System | TNT® T7 Quick Coupled Transcription/Translation System, Trial Size | Transcend™ tRNA |
| 18. |
Grover, G.S., Turner, B.A., Parker, C.N., Meier, J., Lala, D. S., and Lee, P.H.
(2003)
Multiplexing Nuclear Receptors for Agonist Identification in a Cell-Based Reporter Gene High-Throughput Screen.
J. Biomol. Screen.
8
,
239-246
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Notes:
Nuclear receptor ligand binding regions from the Farnesoid-X-activated receptor (FXR) and peroxisome proliferator-activated receptor delta (PPARδ) were fused to a Gal4 DNA Binding Domain. These constructs were used to screen compounds from a LOPAC library of 640 compounds. Ligands bound to the Nuclear receptor ligand binding regions would result in the fusion proteins binding and inducing expression of a luciferase reporter construct with 5 upstream Gal4 binding element repeat sequences. For these experiments, Human hepatoma cell line (Huh7) cells transiently transfected with both plasmids were seeded in 384 well plates. After exposure to the various compounds in the LOPAC library luciferase expression was assessed with the Steady-Glo® Luciferase Assay System. Cells and Steady-Glo® reagent were added to cultures by a Labsystem Multidrop-384 liquid dispenser. Plates were read on a Molecular Devices ChemiLuminescence Imaging Plate Reader.
(0003265) |
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Products: Steady-Glo® Luciferase Assay System |
| 19. |
Rapisarda, A., Uranchimeg, B., Scudiero, D.A., Selby, M., Sausville, E.A., Shoemaker, R.H. and Melillo, G.
(2002)
Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway.
Cancer Res.
62(15)
,
4316-4324
.
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Notes:
The authors of the study undertook a screen of small molecules to find chemicals that reduce gene expression caused by hypoxia. Neomycin resistant stable cell lines derived from U251 human glioma cells expressing luciferase under the control of either the HSV thymidine kinase (TK) with or without hypoxia-response element (HRE) repeats, or the SV40 promoter were constructed. U251 cell lines expressing luciferase under control of either a vascular endothelial growth factor (VEGF) promoter or a minimal HRE containing VEGF promoter were also tested. To screen for inhibitors of the hypoxia induced pathway, 3,000 cells in 25µl/well were plated in 384-well plates using a Beckman Coulter Biomek® 2000 automated workstation. Cells were cultured for 24 hours under normal conditions, then compounds from the NCI "Diversity Set" of approximately 2,000 compounds were added (in 25µl of carrier), and the cells were placed in a hypoxia chamber for 20 hours. Cells were allowed to recover for 1.5 hours in normoxic conditions before addition of 40µl/well Bright-Glo™ Luciferase Assay Reagent. Luminescence was recorded using a Tecan Ultra Multifunction Plate Reader in luminescence mode. Further analyses of compound "hits" were performed on the VEGF-promoted luciferase cell lines in 96-well plates.
(0003096) |
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Products: Bright-Glo™ Luciferase Assay System | pGL2-Basic Vector | pGL2-Promoter Vector | pGL3-Control Vector |
| 20. |
Hirose, F., Ohshima, N., Kwon, E.-J., Yoshida, H., and Yamaguchi, M.
(2002)
Drosophila Mi-2 Negatively Regulates dDREF by Inhibiting Its DNA-Binding Activity.
Mol. Cell. Biol.
22
,
5182-5193
.
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Notes:
Drosophila Mi-2 was discovered to specifically bind the DNA binding domain of dDREF (DNA replication-related element factor) by yeast two hybrid screen. This binding was shown to inhibit dDREF’s transcriptional regulatory action on a DNA replication-related element (DRE). Analysis of dDREF binding to the DRE was performed by examination of expression from –168DPCNAluc, –168mutΔPCNAluc, and pRL-actin5C reporter genes in Schneider cells using the Dual-Glo™ Luciferase Assay System.
(0002622) |
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Products: Dual-Glo® Luciferase Assay System | Luciferase Assay System |
| 21. |
Contanzo, A., Guiet, C., Vito, P.
(1999)
c-E10 is a caspase-recruiting domain-containing protein that interacts with components of death receptors signaling pathway and activates nuclear factor-κB
J. Biol. Chem.
274
,
20127-20132
.
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Notes:
The authors identified c-E10, a protein that has an N-terminal caspase-recruiting domain, in a screen for molecules involved in regulation of death receptor signaling pathways. A cDNA encoding viral-E10 was transcribed and translated in vitro using Promega TNT Coupled Reticulocyte Lysate Systems. Additionally, HeLa cells were transfected with a variety of cDNAs together with a β-galactosidase reporter gene, treated with TNFα, and then assayed for cell death using the CellTiter® Non-Radioactive Cell Proliferation Assay. Expression of c-E10 did not affect the ability of TNFα to induce apoptosis in the transfected cells. The authors also used the Luciferase Assay System to assess NF-κB activation in the transfected cells.
(0002502) |
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Products: CellTiter 96® Non-Radioactive Cell Proliferation Assay | Luciferase Assay System | TNT® SP6 Coupled Reticulocyte Lysate System | TNT® T3 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System | TNT® T7/SP6 Coupled Reticulocyte Lysate System | TNT® T7/T3 Coupled Reticulocyte Lysate System |