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| 1. |
Wilson, P.M., Fazzone, W., LaBonte, M.J., Lenz, H.J. and Ladner, R.D.
(2009)
Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage.
Nucleic Acids Res.
37
,
78–95
.
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Notes:
The authors examined the role of p53 in modulating dUTPase promoter activity. Base substitution mutations of Sp1- and E2F-binding sites in the dUTPase promoter were performed using the GeneEditor™ in vitro Site-Directed Mutagenesis System. Each mutant was confirmed by DNA sequencing. To determine growth inhibition, HCT116 human colon cancer cells were seeded in 96-well plates at 3 × 103 cells/well and treated with 5-fluorouracil (5-FU), fluorodeoxyuridine (FUdR), oxaliplatin or in combination. After 72 hours, the CellTiter® 96 AQueous One Solution was dispensed into each well and absorbance measured. RNA was isolated from HCT116 p53+/+ and HCT116 p53-/- cells. cDNA was reverse transcribed from 200ng total RNA followed by multiplex qPCR using the Plexor™ qPCR System to amplify dUTPase, thymidylate synthase and GAPDH, a housekeeping gene. The 1.2 kb region of the dUTPase promoter upstream of the transcriptional start site was amplified by PCR and the fragment cloned into the pGL3-Basic Vector. Truncated promoters were also generated by PCR and cloned into the same vector. Drosophila SL-2 cells and HCT116 cell lines were seeded in a 24-well plate and transfected with dUTPase pGL3 promoter constructs or with pCI-Neo:p53WT, pCI-Neo:p53MUT and the empty pCI-neo Mammalian Expression Vector; all transfections included the pRL-TK Vector at a ratio of 1:10. After six hours, the cells were incubated in either fresh medium or medium containing a cytotoxic agent at the appropriate concentration. Thirty hours later, the cells were lysed, quantitated by Western blotting and 20µl of lysate analyzed with the Dual-Luciferase® Reporter Assay System. Electrophoretic mobility shift analyses (EMSA) were performed using –64 to –91 of the dUTPase-nuclear isoform transcriptional start site in the Gel Shift Assay System.
(0004031) |
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Products: CellTiter 96® AQueous One Solution Cell Proliferation Assay | CellTiter 96® AQueous One Solution Reagent | CellTiter 96® AQueous One Solution Reagent | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | Gel Shift Assay Core System | Gel Shift Assay System | GeneEditor™ in vitro Site-Directed Mutagenesis System | pCI Mammalian Expression Vector | pGL3 Basic Vector | Plexor® qPCR System | pRL-TK Vector |
| 2. |
Zheng, Y., Ritzenthaler, J.D., Sun, X., Roman, J. and Han, S.
(2009)
Prostaglandin E2 stimulates human lung carcinoma cell growth through induction of integrin-linked kinase: the involvement of EP4 and Sp1.
Cancer Res.
69
,
896–904
.
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Notes:
In this paper, the role of prostaglandin E2 (PGE2) stimulation of integrin-linked kinase (ILK) in human lung carcinoma was explored. Mutations of Sp1 and NF-κB cis-acting elements in an ILK promoter-pGL3-Basic Vector construct were created using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The mutations were confirmed via sequencing. Human non–small cell lung carcinoma (NSCLC) cells were plated at a density of 5 × 105 cells per well in six-well plates and transfected with 2µg of ILK promoter reporter vectors with or without 0.2µg of the phRL-TK Renilla Luciferase Reporter Vector. After 24 hours, the transfected cells were exposed to PGE2 and the cells lysed for assessment using the Dual-Luciferase® Reporter Assay System. NSCLC cells were transfected with inactive (ILK-S343A) and superactive ILK (ILK-S343D) cDNA, incubated for 24 hours, treated with or without exogenous PGE2 or with an Sp1 inhibitor for 2 hours. The numbers of viable cells were measured using the CellTiter-Glo® Luminescent Cell Viability Assay.
(0004026) |
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Products: CellTiter-Glo® Luminescent Cell Viability Assay | Dual-Glo® Luciferase Assay System | GeneEditor™ in vitro Site-Directed Mutagenesis System | pGL3 Basic Vector |
| 3. |
Reisman, S.A,, Yeager, R.L., Yamamoto, M. and Klaassen, C.D.
(2009)
Increased Nrf2 activation in livers from Keap1-knockdown mice increases expression of cytoprotective genes that detoxify electrophiles more than those that detoxify reactive oxygen species.
Toxicol. Sci.
108
,
35–47
.
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Notes:
In this study, the researchers wanted to determine the role of kelch-like ECH associated protein 1 knockdown (Keap1-kd) mice protein products, which are thought to protect against oxidative and electrophilic stress, and compare the hepatic phenotype with that of transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2)-null and wild-type mice. Microsomal suspensions from liver homogenates were prepared, and bile was collected from wild-type, Nrf2-null, and Keap1-kd mice. Reduced GSH was quantified using the GSH-Glo™ Glutathione Assay.
(0004012) |
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Products: GSH-Glo™ Glutathione Assay |
| 4. |
Cho, Y.Y., Tang, F., Yao, K., Lu, C., Zhu, F., Zheng, D., Pugliese, A., Bode, A.M. and Dong, Z.
(2009)
Cyclin-dependent kinase-3-mediated c-Jun phosphorylation at Ser63 and Ser73 enhances cell transformation.
Cancer Res.
69
,
272–281
.
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Notes:
This paper examined the role of the Cdk3/c-Jun signaling in EGF-stimulated cell proliferation and cell transformation. An AP-1 luciferase reporter plasmid construct was contransfected with various expression vector combinations of Cdk3, Cdk2, c-Jun, c-Jun M63/73, Cdk3-DN and cyclin C and the phRL-SV40 Vector as a normalization control. Cells were lysed at room temperature for 30 minutes with gentle shaking and luciferase activity measured using the Dual-Luciferase® Reporter Assay System. SaoS-2 cells transfected with a mock and Cdk3 RNAi vector were seeded at a density of 4 x 103 in 96-well plates with 20µl of medium. After 24 hours, 20µl of CellTiter® 96 AQueous One Solution was dispensed to each well, and the plate incubated for 1 hour at 37°C. The reaction was stopped using 25µl of 10% SDS and absorbance was measured at 492 and 690nm. For the CheckMate™ Mammalian Two-Hybrid System, the plasmid constructs, pACT-c-Jun, pBIND-Cdk3 and pG5luc, were cotransfected into HEK293 cells at no more than 100ng/well in a 48-well plate. After incubation and lysis, 20µl of lysate was dispensed into each well of a 96-well luminescence plate. Firefly and Renilla luciferase activity was detected.
(0004028) |
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Products: CellTiter 96® AQueous One Solution Cell Proliferation Assay | CheckMate™ Mammalian Two-Hybrid System | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pACT Vector | pBIND Vector | pG5luc Vector |
| 5. |
Wu, Y., Connors, D., Barber, L., Jayachandra, S., Hanumegowda, U.M. and Adams, S.P.
(2009)
Multiplexed assay panel of cytotoxicity in HK-2 cells for detection of renal proximal tubule injury potential of compounds
Toxicology in Vitro
23
,
1170-1171
.
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Notes:
The authors describe a multiplexed in vitro assay to detect nephrotoxicity and gain information about mechanism of cell death in HK-2 (human kidney-2) cells. The multiplexed assay involved an LDH assay to detect necrosis, a caspase-3/7 assay to detect apoptosis, a reazurin assay to assess metabolic state, and a DNA dye staining assay to monitor nuclear morphology.
(0004002) |
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Products: Caspase-Glo® 3/7 Assay | Caspase-Glo® 3/7 Buffer | Caspase-Glo® 3/7 Substrate | CellTiter-Blue® Cell Viability Assay | CellTiter-Glo® Buffer | CellTiter-Glo® Luminescent Cell Viability Assay | CellTiter-Glo® Substrate (lyophilized) | CytoTox-ONE™ Homogeneous Membrane Integrity Assay | CytoTox-ONE™ Homogeneous Membrane Integrity Assay, HTP |
| 6. |
Niles, A.L., Moravec, R.A. and Riss, T.L.
(2009)
In vitro viability and cytotoxicity testing and same-well multi-parametric combinations for high-throughput screening
Current Chemical Genomics
3
,
33-41
.
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Notes:
The authors review the use of in vitro cytotoxicity testing in drug discovery to characterize the toxic potential of new chemical entities (nce) at the earliest stages of profiling. DOI: 10.2174/1875397300903010033
(0004000) |
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Products: Apo-ONE® Homogeneous Caspase-3/7 Assay | Apo-ONE® Homogeneous Caspase-3/7 Buffer | Caspase-Glo® 3/7 Assay | CellTiter-Blue® Cell Viability Assay | CellTiter-Fluor™ Cell Viability Assay | CellTiter-Glo® Assay Custom Solution | CellTiter-Glo® Luminescent Cell Viability Assay | MultiTox-Fluor Multiplex Cytotoxicity Assay | MultiTox-Glo Multiplex Cytotoxicity Assay |
| 7. |
Gupta, P.B., Onder, T.T., Jiang, G., Tao, K., Kuperwasser, C., Weinberg, R.A. and Lander, E.S.
(2009)
Identification of selective inhibitors of cancer stem by high-throughput screening.
Cell
138
,
645-59
.
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Notes:
The authors of this study describe a proof-of-concept screen to use mammary epithelial cells that have been induced to undergo an epithelial to mesenchymal transition (EMT) as model cells to identify agents that may be selectively toxic against "epithelial cancer stem cells" (CSCs). They induced the transformed breast cancer cell line HMLER to undergo a mesenchymal transition using shRNA directed against the E-cadherin gene. They characterized the responsiveness of these transitioned cells to common cytotoxic agents using the CellTiter® 96 AQueous Cytoxicity Assay and compared the response to that of HMLER cells containing a control shRNA. They showed that the HMLER cells induced to undergo EMT behaved more like CSCs. The researchers then performed a proof-of-concept high-throughput screen to identify compounds that targeted the HMLER cells induced to undergo EMT, using the CellTiter-Glo® Assay to assess cell viability.
(0004006) |
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Products: CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay | CellTiter-Glo® Assay Custom Solution | CellTiter-Glo® Buffer | CellTiter-Glo® Luminescent Cell Viability Assay | CellTiter-Glo® Substrate (lyophilized) |
| 8. |
Wong, D.J., Nuyten, D.S.A., Regev, A., Lin, M., Adler, A.S., Segal, E., van de Vijver, M.J. and Chang, H.J.
(2008)
Revealing targeted therapy for human cancer by gene module maps
Cancer Res.
68
,
369-378
.
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Notes:
The authors of this study used the gene module map method to identify genotype-specific therapies for human cancers. They studied breast cancer cell lines that expressed the "mitochondria" and "wound signatures". These cell lines, which are associated with tumors that have poor prognosis, were also shown to have a "proteasome signature" and were sensitive to the proteasome inhibitor bortezomib. The authors used the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay to show that genotype-specific killing of "wound signature" cells was not due to differential degrees of proteasome inhibition, since 1 and 1µmol/L of bortezomib completely inhibited the ability of the wound signature and control cells to cleave the luminogenic substrate. More likely, the wound signature genotype conferred increased susceptibility to death from proteasome inhibition.
(0003870) |
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Products: Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay |
| 9. |
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 |
| 10. |
Grenier, A.L., Abu-Ihweij, K., Zhang, G., Ruppert, S.M., Boohaker, R., Slepkov, E.R., Pridemore, K., Ren, J.J., Fliegel, L. and Khaled, A.R.
(2008)
Apoptosis-induced alkalinization by the Na+/H+ exchanger isoform 1 is mediated through phosphorylation of amino
Am. J. Physiol. Cell Physiol.
295
,
C883-C896
.
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Notes:
The authors wanted to examine the role of plasma membrane protein Na+/H+ exchanger isoform 1 (NHE1) in apoptosis. API cells, a NHE1-deficient Chinese hamster ovary cell line, was cotransfected with wild-type NHE1 or mutant NHE1 constructs and destabilized yellow fluorescent protein (YFP). Cells were plated at a density of 1 × 104 cells/well in a 96-well plate with or without FBS. To induce apoptosis in the cells, serum was withdrawn for 24 hours. The ratio of dead-to-live cells was measured using the MultiTox-Fluor Multiplex Cytotoxicity Assay. Cell death was also determined by examining the loss of YFP fluorescence under a microscope.
(0003937) |
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Products: MultiTox-Fluor Multiplex Cytotoxicity Assay |
| 11. |
Cali, J.J., Niles, A., Valley, M.P., O’Brien, M.A., Riss, T.L., and Shultz, J.
(2008)
Bioluminescent assays for ADMET
Expert Opin. Drug Metab. Toxicol.
4
,
103–120
.
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Notes:
The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase.
(0003926) |
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Products: Apo-ONE® Homogeneous Caspase-3/7 Assay | Apo-ONE® Homogeneous Caspase-3/7 Buffer | Beta-Glo® Assay System | Calpain-Glo™ Protease Assay | Caspase-Glo® 2 Assay | Caspase-Glo® 3/7 Assay | Caspase-Glo® 6 Assay | Caspase-Glo® 8 Assay | Caspase-Glo® 9 Assay | CellTiter-Glo® Luminescent Cell Viability Assay | GloResponse™ CRE-luc2P HEK293 Cell Line | GloResponse™ NFAT-RE-luc2P HEK293 Cell Line | GSH-Glo™ Glutathione Assay | Kinase-Glo® Luminescent Kinase Assay | Kinase-Glo® Max Luminescent Kinase Assay | Kinas |
| 12. |
Zheng, D., Cho, Y.Y., Lau, A.T., Zhang, J., Ma, W.Y., Bode, A.M. and Dong, Z.
(2008)
Cyclin-dependent kinase 3-mediated activating transcription factor 1 phosphorylation enhances cell transformation.
Cancer Res.
68
,
7650–7660
.
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Notes:
To examine the role of cyclin-dependent kinase (cdk)-3 expression in cancer cell lines, potential targets of cdk3 phosphorylation were examined. The CheckMate™ Mammalian Two-Hybrid System was used to test for transcription factor binding partners of cdk in HEK293 cells. Cell proliferation of T96G cells stably transfected with either cdk3 or vector was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay.
(0003983) |
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Products: CellTiter 96® AQueous One Solution Cell Proliferation Assay | CheckMate™ Mammalian Two-Hybrid System |
| 13. |
Hahn, C.K., Ross, K.N., Warrington, I.M., Mazitschek, R., Kanegai, C.M., Wright, R.D., Kung, A.L., Golub, T.R. and Stegmaier, K.
(2008)
Expression-based screening identifies the combination of histone deacetylase inhibitors and retinoids for neuroblastoma differentiation.
Proc. Natl. Acad. Sci. USA
105
,
9751-9756
.
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Notes:
The authors designed a high-throughput gene-expression screen to identify compounds that induce a neuroblastoma gene signature in BE(2)-C cells. The screen used Valproic Acid (VPA), an inhibitor of histone deacetylase, as an enhancer. The top hit from the screen was all-trans retinoic acid (ATRA). The authors proposed that ATRA + VPA would reduce cell viability and might induce apoptosis by inducing genes associated with terminal differentiation. They used the CellTiter-Glo® Luminescent Cell Viability Assay in a 96-well format to assess the effects of ATRA and a variety of inhibitors of histone deacetylase on BE(2)-C cell viability.
(0003931) |
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Products: CellTiter-Glo® Luminescent Cell Viability Assay |
| 14. |
Lin, H., Lee, E., Hestir, K., Leo, C., Huang, M., Bosch, E., Halenbeck, R., Wu, G., Zhou, A., Behrens, D., Hollenboguh, D., Linnemann, T., Qin, M., Wong, J., Chu, K., Doberstein, S.K. and Williams, L.T.
(2008)
Discovery of a cytokine and its receptor by functional screening of the extracellular proteome.
Science
320
,
807-11
.
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Notes:
The authors of this study created a cDNA library representative of the extracellular proteome (secreted proteins and the extracellular domains of transmembrane proteins). Each cDNA was individually transfected into 293T cells. Medium from the cDNA of each transfection was used in a suite of cell-based assays. The CellTiter-Glo® Assay was used to screen for secreted factors from the cell lines expressing the cDNA that affected viability of twelve cell lines: human primary B cells, human primary T cells, human primary NK cells, human primary monocytes, A549 cells, Colo205 cells, U-118 cells, MDA-MB231 cells, PC3 cells, PANC1 cells, human primary skeletal muscle progenitor cells, and rat primary oligodendrocyte precursor cells.
(0003935) |
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Products: CellTiter-Glo® Luminescent Cell Viability Assay |
| 15. |
Case, M., Matheson, E., Minto, L., Hassan, R., Harrison, C.J., Bown, N., Bailey, S., Vormoor, J., Hall, A.G. and Irving, J.A.E.
(2008)
Mutation of genes affecting the RAS pathway is common in childhood acute lymphoblastic leukemia
Cancer Res.
68
,
6803-6809
.
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Notes:
The authors of this study investigated the relationship of somatic mutations that deregulate the RAS-RAF-MEK-ERK pathway and Acute Lymphoblastic Leukemia (ALL) and its progression to relapse in some patients. They show that such mutations are common in ALL and its recurrence. Furthermore, lymphoblasts from patients with mutations that were associated with upregulation of ERK showed increased cytotoxicity over wild-type controls when treated with U0126, suggesting that specific ERK inhibitors may eventually yield useful therapeutics. Following drug exposure, cytotoxic effects were assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay.
(0003905) |
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Products: CellTiter 96® AQueous One Solution Cell Proliferation Assay | MEK Inhibitor U0126 |
| 16. |
Jin, Z., Lahat, G., Korchin, B., Nguyen, T., Zhu, Q.S., Wang, X., Lazar, A.J., Trent, J., Pollock, R.E. and Lev D.
(2008)
Midkine enhances soft-tissue sarcoma growth: a possible novel therapeutic target.
Clin. Cancer Res.
14
,
5033–42
.
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Notes:
Increased expression of midkine (MK), a growth factor normally involved in neural development, is associated with several human malignancies. The authors used quantitative RT-PCR to examine mRNA levels for MK and MK receptors in various soft-tissue sarcoma (STS) cell lines. Reverse transcription was performed using 1µg of total RNA, and 2µl of cDNA was used in qPCR using the PCR Master Mix, primers specific to MK and either glyceraldehyde-3-phosphate or β-actin primers for normalization, and EvaGreen® dye. To examine the protumorigenic effects of MK, the authors incubated HT1080 and SW684 STS cell lines, which express low levels of MK, with human recombinant MK and measured cell proliferation using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. In addition, the authors examined cell proliferation in MK-stably transfected HT1080 cells. The plasmid used for stable transfections was created by reverse transcribing the MK-coding region using the ImProm-II™ Reverse Transcription System, then cloning the resulting cDNA into an expression vector.
(0003895) |
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Products: CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay |
| 17. |
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 |
| 18. |
Prichard, M.N., Daily, S.L., Jefferson, G.M., Perry, A.L. and Kern, E.R.
(2007)
A rapid DNA hybridization assay for the evaluation of antiviral compounds against Epstein-Barr virus.
J. Virol. Methods.
144
,
86–90
.
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Notes:
The authors developed an assay to evaluate antiviral compounds for Epstein-Barr virus (EBV) infections. EBV DNA was isolated using the Wizard® SV 96 Genomic DNA Purification System, and 5µl was used for real-time PCR to quantify the viral DNA. Cytotoxicity of the antiviral compounds was assessed using treated, uninfected Akata cells compared to those with no treatment or infection. After three days when the viral DNA was harvested, cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay.
(0003761) |
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Products: CellTiter-Glo® Luminescent Cell Viability Assay | Wizard® SV 96 Genomic DNA Purification System |
| 19. |
Wu, D., Ren, Z., Pae, M., Guo, W., Cui, X., Merrill, A.H. and Meydani, S.N.
(2007)
Aging up-regulates expression of inflammatory mediators in mouse adipose tissue.
J. Immunol.
179
,
4829–4839
.
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Notes:
The authors examined the role of ceramide and NF-κB in age-related adipose tissue inflammation in mice. Levels of inflammation-associated molecules, IL-1β, IL-6, TNF-α, COX-2 and peroxisome proliferator-activated receptor (PPAR)-γ mRNA, were quantified by quantitive PCR (qPCR). RNA was isolated from adipose tissues, and first-strand cDNA was synthesized using the Reverse Transcription System prior to qPCR. mRNA levels were also quantified in peritoneal macrophages grown in the presence of young adipocyte-conditioned medium and old adipocyte-conditioned medium. Viability of the primary adipocytes used in these experiments was confirmed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay and CytoTox 96® Non-Radioactive Cytotoxicity Assay.
(0003777) |
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Products: CellTiter 96® AQueous One Solution Cell Proliferation Assay | CytoTox 96® Non-Radioactive Cytotoxicity Assay | Reverse Transcription System |
| 20. |
Treeck, O., Pfeiler ,G., Horn, F., Federhofer, B., Houlihan, H., Vollmer, A., and Ortmann, O.
(2007)
Novel estrogen receptor beta transcript variants identified in human breast cancer cells affect cell growth and apoptosis of COS-1 cells.
Mol. Cell. Endocrinol.
264
,
50-60
.
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Notes:
This study identified two novel transcript variants of the estrogen receptor ERβ that were expressed in the ERα-negative breast cancer cell line MDA-MD-231. These variants were identified after amplification of ERβ transcripts from the breast cancer cell line by RT-PCR. The amplification products were then excised from gels and subcloned into the pTARGET™ Mammalian Expression Vector prior to sequencing. COS1 cells, which do not express the estrogen receptor, were then stably transfected with full-length ERβ or one of the splice variants and the effects on cell proliferation, apoptosis, and estrogen response were evaluated. In COS1 cells expressing either ERβ or the transcript variants cell proliferation decreased and basal apoptosis (caspase 3/7 activity) increased, compared to cells transfected with vector alone. Exposure to therapeutic doses of tamoxifen induced apoptosis in cells expressing the full-length ERβ but not in cells expressing either of the variant isoforms.
(0003618) |
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Products: Caspase-Glo® 3/7 Assay | CellTiter-Blue® Cell Viability Assay | ImProm-II™ Reverse Transcriptase | M-MLV Reverse Transcriptase Buffer Pack | M-MLV Reverse Transcriptase, RNase H Minus | pTARGET™ Mammalian Expression Vector System |
| 21. |
Garcia-Pedrero, J.M, Kiskinis, E., Parker, M.G., and Belandia, B.
(2007)
The SWI/SNF chromatin remodeling subunit BAF57 is a critical regulator of estrogen receptor function in breast cancer cells.
J. Biol. Chem.
281
,
22656–22664
.
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Notes:
To examine the role that BAF57, a transcriptional modulator of the estrogen receptor (ER), may have in breast cancer, BT549 cells were transfected with a reporter vector (pGL3-Basic with two estrogen response elements and the human pS2 promoter), the control pRL-CMV Vector and combinations of the following expression vectors: mERα or hERβ, BAF57, SRC1e, SRC1a and RAC3. After 16 hours, the cells were treated with 17β-estradiol. Twenty-four hours later, the cells were harvested and the luciferase activities assayed using the Dual-Luciferase® Reporter Assay System. GST-BAF57 (full-length, N- or C-domain) fusion protein was bound to Sepharose beads and incubated with 17β-estradiol or vehicle and wildtype or one of various mERα interaction domain mutants, which have been expressed and labeled with 35S methionine using a TNT® rabbit reticulocyte lysate system. The beads were washed and analyzed for bound protein. ZR-75-1 cells were transfected with BAF57 siRNA then treated with 17β-estradiol 24 hours later. Cell proliferation was measured using the CellTiter® 96 AQueous One Solution Cell Proliferation Assay.
(0003599) |
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Products: CellTiter 96® AQueous One Solution Cell Proliferation Assay | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL3-Basic Vector | pRL-CMV Vector |
| 22. |
Zhuang, H. and Matsunami, H.
(2007)
Synergism of accessory factors in functional expression of mammalian odorant receptors.
J. Biol. Chem.
282
,
15284–15293
.
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Notes:
To explore the role that accessory proteins may play in successfully expressing odorant receptors (ORs) on the cell surface of heterologous cells, the authors explored cotransfecting receptor-transporting proteins RTP1S, Ric8b and Gαolf with an N-terminally tagged OR to generate a functional and ligand-specific expression system. Three tags (Rho, FLAG or HA) were inserted into the NheI and EcoRI sites of the pCI Mammalian Expression Vector. Amplified OR orfs were cloned into the MluI and NotI sites of the tagged pCI Mammalian Expression Vector. The accessory proteins were subcloned into HA-pCI (RTP1S) or pCI (RTP1S, Ric8b, Hsc70t and Gαolf). For cotransfection, the N-terminal tagged OR vectors (0.8µg) and the assessory proteins (individually or in combination; 0.8µg) were transfected into HEK293T or Hana3A cells. The transfected cells were then subjected to immunocytochemistry, live-cell surface staining, permeabilized staining or FACS analysis. The Dual-Glo™ Luciferase Assay System was used to assess OR activation via CRE elements on a firefly luciferase vector. pRL-SV40 Vector was the internal control for cell viability and transfection efficiency. HEK293T or Hana3A cells were plated on 96-well plates, transfected with 1µg of CRE-Luc vector, 1µg of pRL-SV40 Vector, 5µg of OR and 1µg total for all accessory proteins (0.25µg each protein with pCI Mammalian Expression Vector to keep amount of plasmid constant). Twenty-four hours posttransfection, the medium was changed to CD293 chemically defined medium, incubated for 30 minutes at 37°C then replaced with 25µl of odorant solution in CD293 for a second incubation of 4 hours at 4°C. Then the reporter protein expression levels were measured by luminescence.
(0003687) |
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Products: Dual-Glo® Luciferase Assay System | pCI Mammalian Expression Vector | pRL-SV40 Vector |
| 23. |
Niles, A.L., Moravec, R.A., Hesselberth, P.E., Scurria, M.A., Daily, W.J. and Riss, T.L.
(2007)
A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers
Anal. Biochem.
366
,
197–206
.
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Notes:
The authors of this paper describe an assay that uses protease biomarkers to assess cell viability and cell death simultaneously in a population of cells. The assay detects an ubiquitous protease activity that is associated with live cells and a second protease activity that is associated with cells that have lost membrane integrity. The readouts are either fluorescent or fluorescent and luminescent. The assay can be performed in multiplex with other assays, such as caspase assays, to gain additional information on the cell population, and it is amenable to high-throughput screening.
(0003927) |
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Products: CytoTox-Glo™ Cytotoxicity Assay | MultiTox-Fluor Multiplex Cytotoxicity Assay | MultiTox-Glo Multiplex Cytotoxicity Assay |
| 24. |
Lynch, R.A., Etchin, J., Battle, T.E. and Frank, D.A.
(2007)
A small-molecule enhancer of signal transducer and activator of transcription 1 transcriptional activity accentuates the antiproliferative effects of IFN-γ in human cancer cells
Cancer Res.
67
,
1254-1261
.
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Notes:
STAT1 is a transcription factor that is involved in a variety of cellular processes and behaves like a tumor suppressor in many ways. It is associated with apoptosis, inhibition of cyclin-dependent kinases, and it may mediate the antitumor effects of IFN-γ. The authors of this study designed a bioluminescent reporter assay to identify small molecules that enhance STAT1-dependent gene expression. The bioluminescent assay was performed in 384-well plates using both stably and transiently transfected cell lines. The screening assay was robust, producing a Z´factor value of 0.92, and it identified three compounds that specifically enhanced STAT1-dependent transcriptional activity. Firefly luciferase activity in stable cell lines was assessed using the Bright-Glo® Luciferase Assay System; luciferase activity in the transiently transfected cells was monitored using the Dual-Luciferase® Assay System. Viability of cells in culture was monitored using the CellTiter-Glo® Assay.
(0003732) |
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Products: Bright-Glo™ Luciferase Assay System | CellTiter-Glo® Luminescent Cell Viability Assay | Dual-Luciferase® Reporter Assay System | pRL-TK Vector |
| 25. |
Fan, F. and Wood, K.V.
(2007)
Bioluminescent assays for high-throughput screening
Assay Drug Dev. Technol.
5
,
127–136
.
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Notes:
The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays).
(0003737) |
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Products: Renilla Luciferase Assay System | BacTiter-Glo™ Microbial Cell Viability Assay | Bright-Glo™ Luciferase Assay System | Calpain-Glo™ Protease Assay | cAMP-Glo™ Assay | Caspase-Glo® 2 Assay | Caspase-Glo® 3/7 Assay | Caspase-Glo® 6 Assay | Caspase-Glo® 8 Assay | Caspase-Glo® 9 Assay | CellTiter-Glo® Luminescent Cell Viability Assay | Dual-Glo® Luciferase Assay System | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | Kinase-Glo® Lumine |