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| 1. |
Hornakova, T., Staerk, J., Royer, Y., Flex, E., Tartaglia, M., Constantinescu, S.N., Knoops, L. and Renauld, J.C.
(2009)
Acute lymphoblastic leukemia-associated JAK1 mutants activate the Janus kinase/STAT pathway via interleukin-9 receptor alpha homodimers.
J. Biol. Chem.
284
,
6773–6781
.
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Notes:
The authors studied the ability of JAK1 V658F and A634D mutants to activate the Janus kinase (JAK)/STAT pathway when expressed alone or together with the other components of the interleukin-9 receptor complex. The BOX1 motif of wild-type IL-9Rα, the JAK interacting region, was mutated from PXP to SXS using the GeneEditor™ in vitro Site-Directed Mutagenesis System. To assess STAT transcriptional activity, HEK293 human embryonic kidney, COS-7 monkey kidney, U4C human fibrosarcoma and g2A cells were cotransfected with 250ng of the appropriate constructs, 500ng of firefly luciferase vectors and 50ng of pRL-TK Vector and empty plasmid for a total 1.5µg of DNA. After 24 hours, the cells were lysed in 150µl of Passive Lysis Buffer and reporter activity measured using the Dual-Luciferase® Reporter Assay System. The ProFection® Mammalian Transfection System—Calcium Phosphate was used to transfect 106 HEK293 cells in a six-well plate with 3.75µg of plasmid for Western blot analysis and cotransfected 6 × 106 HEK293 cells in a 100mm dish with 14µg plasmid for immunoprecipitation studies.
(0004025) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | GeneEditor™ in vitro Site-Directed Mutagenesis System | pRL-TK Vector | ProFection® Mammalian Transfection System—Calcium Phosphate |
| 2. |
Wruck, C.J., Götz, M.E., Herdegen, T., Varoga, D., Brandenburg, L-O. and Pufe, T.
(2008)
Kavalactones protect neural cells against amyloid β peptide-induced neurotoxicity via ERK1/2-dependent Nrf2-activation
Molecular Pharmacology Fast Forward
March 11, 2008
,
epub ahead of print
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Notes:
The accumulation of the toxic form of the Amyloid-β peptide is known to induce oxidative damage in the brain. Although treatment with antioxidants has not proven effective at controlling AD symptoms, inducing the natural systems in the brain that protect from oxidative damage may provide a possible therapeutic approach. A host of antioxidant and detoxifying enzymes are upregulated by binding of the Nrf2 transcription factor to the ARE (antioxidant response element) regulatory sequence. The authors used a Dual Luciferase® Reporter Assay to assess modulation of gene activity through ARE by kavalactones. Kavalactones are compounds found in the roots and rhizomes of Kava (Piper methysticum), a plant cultivated an used in some Pacific societies for medicinal and social uses. The ARE1 region from the rat NAD(P)H:quinone oxidoreductase-1 gene was placed upstream of a pGL3 firefly luciferase reporter construct and cotransfected along with a pRL-TK Renilla control construct into PC12 or C6 cells. The data show induction of luciferase activity by kavalactones. Further investigation shows that the kavalactones promote Nrf2 stabilization possibly through the ERK1/2 pathway.
(0003859) |
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Products: Dual-Luciferase® Reporter Assay System | pGL3-Basic Vector | pGL3-Control Vector | pGL3-Enhancer Vector | pGL3-Promoter Vector | pRL-TK Vector |
| 3. |
Gallmeier, E., Hucl, T., Brody, J.R., Dezentje, D.A., Tahir, K., Kasparkova, J., Brabec, V., Bachman, K.E. and Kern, S.E.
(2007)
High-throughput screening identifies novel agents eliciting hypersensitivity in Fanconi pathway-deficient cancer cells.
Cancer Res.
67
,
2169–2177
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Notes:
The Fanconi anemia (FA) pathway is inactivated in a variety of human tumors. Identifying novel compounds that affect FA-pathway deficient cells could provide further information on the FA pathway as well as new therapeutic possibilities. In this study, RKO cells, a human cancer cell line stably expressing firefly luciferase under the control of the p53 consensus DNA-binding site, were treated with 10 and 20µmol/L 80136342, a new compound that enhances cancer cell survival, or 50µmol/L etoposide, a known activator of p53, for 24 hours. The reporter activity was assessed using the Steady-Glo® Luciferase Assay System and compared to that seen in untreated controls.
(0003600) |
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Products: Steady-Glo® Luciferase Assay System |
| 4. |
Su, H.W., Yeh, H.H., Wang, S.W., Shen, M.R., Chen, T.L., Kiela, P.R., Ghishan, F.K. and Tang, M.J
(2007)
Cell confluence-induced activation of signal transducer and activator of transcription-3 (Stat3) triggers epithelial dome formation via augmentation of sodium hydrogen exchanger-3 (NHE3) expression.
J. Biol. Chem.
282
,
9883–94
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Notes:
The authors tested their hypothesis that Na+-H+ exchangers (NHE) are involved in the formation of multicullar dome structures in confluent Madin-Darby canine kidney (MDCK) cells and that the Stat3 pathway is involved in regulation of NHEs. The authors performed semi-quantitative RT-PCR to monitor NHE3 mRNA levels in MDCK cells expressing a constitutive Stat3 mutant or a dominant-negative Stat3 mutant. The reverse transcription step was performed using Promega M-MLV Reverse Transcriptase. RAlso, Stat3 activities in low-density cultures and high-density cultures were compared using a reporter gene assay. Four copies of the Stat3-binding site were cloned upstream of a firefly luciferase reporter gene, and the resulting vector, along with the pRL-TK Vector for normalization, were transfected into MDCK cells. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System.
(0003910) |
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Products: Dual-Luciferase® Reporter Assay System | M-MLV Reverse Transcriptase | pRL-TK Vector |
| 5. |
Shukla, V., Coumoul, X., Cao, L., Wang, R.-H., Xiao, C., Xu, X., Andò, S., Yakar, S., LeRoith, D. and Deng, C.
(2006)
Absence of the full-length breast cancer-associated gene-1 leads to increased expression of insulin-like growth factor signaling axis members.
Cancer Res.
66
,
7151-7157
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Notes:
The authors of this study investigated the influence of BRCA1 on insulin-like growth factor 1 (IGF-1) signaling. In mice lacking full-length BRCA, they observed increased IGF-1 expression as well as changes in expression of other proteins within the IGF-1 signaling pathway including Irs-I. They observed increases in IGF-I and Irs-I expression in mammary tumors from these same mice. To understand better the relationship between BRCA1 and IGF, they transfected a mouse mammary tumor cell line with a small hairpin RNA directed against BRCA1 and showed that Irs-1 mRNA and promoter activity increases. Similar results were observed in human UBR60 cells. Irs-1 promoter activity was assessed using the Dual-Luciferase® Reporter Assay System.
(0003604) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack |
| 6. |
Murakami, A., Shigemori, T. and Ohigashi, H.
(2005)
Zingiberaceous and citrus constituents, 1'-acetoxychavicol acetate, zerumbone, auraptene, and nobiletin, suppress lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 murine macrophages through different modes of action.
J. Nutr.
135
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2987S-2992S
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Notes:
These authors investigated the mechanisms by which anti-carcinogenic compounds derived from Japanese and subtropical vegetables and fruits attenuate LPS-induced COX-2 mRNA expression in RAW264.7 mouse macrophages. Using Western blot analysis, 10µg nuclear or 20µg cytosolic protein fractions isolated from LPS-treated macrophages in the absence or presence of various phytochemicals were stained with several antibodies for signal pathway proteins, including the Anti-ERK1/2 pAb. Further analysis of the MAPK and NF-κB systems was performed using firefly luciferase constructs co-transfected with the control pRL-TK Vector at a 1:1 ratio. Transfected cells were exposed to the plant compound for 12 hours and then to LPS for a further 12 hours. Reporter activity was measured using the Dual-Luciferase® Reporter Assay System. To determine the effect of the phytochemical zerumbone on the kinase reaction, a cell-free kinase assay was performed using Kinase-Glo® Assay System. In this assay, 1.5µl recombinant MAPKAPK-2, 0.2µl recombinant active p38α, 1µl zerumbone and 4µmol/l ATP were incubated for 3 hours at 28°C before addition of an equal volume of Kinase-Glo® Reagent.
(0003333) |
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Products: Anti-ERK 1/2 pAb, Rabbit | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | Kinase-Glo® Luminescent Kinase Assay | pRL-TK Vector |
| 7. |
Lin, X., Ruan, X., Anderson, M.G., McDowell, J.A., Kroeger, P.E., Fesik, S.W. and Shen, Y.
(2005)
siRNA-mediated off-target gene silencing triggered by a 7 nt complementation.
Nucleic Acids Res.
33
,
4527-4535
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Notes:
To identify novel regulators of the HIF-1 pathway, the authors performed a knockdown experiment using a synthetic siRNA library against a number of kinases. Two of their ‘top hits’ directly down-regulated the hif-1a mRNA through a 7nt complementation. They built an siRNA library using an HIF-1 reporter assay. For siRNA library screening, the HIF-1 reporter and a control reporter (pRL-TK Vector, Promega) were transfected into H1299 cells. The Dual-Glo™ Luciferase Assay System was used to analyze cells transfected with an siRNA against HIF-1a as a positive control and an siRNA irrelevant to the HIF-1 pathway as a negative control.
(0003538) |
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| 8. |
Sarkar, D., Lebedeva, I.V., Emdad, L., Kang, D-c., Baldwin, A.S. and Fisher, P.B.
(2004)
Human polynucleotide phosphorylase (hPNPase0ld-35): A potential link between aging and inflammation
Cancer Research
64
,
7473-7478
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Notes:
Human polynucleotide phosphorylase (hPNPaseold-35) was originally identified as a gene that is upregulated during cellular differentiation and senescence. The authors of this study show that overexpression of hPNPaseold-35 results in the increased production reactive oxygen species (ROS) and the activation of the NF-κB pathway, resulting in expression of the inflammatory cytokines IL-6 and IL-8. HeLa cells were transfected with either empty pGL3-Basic or 3kB-Luc (pGL3-Basic containing three tandem NF-κB binding sites). Transfected cells were infected with an empty adenoviral vector or one containing hPNPaseold-35. Luciferase assays were conducted using the Luciferase Assay System, and signal was normalized to a pSV-βgal control. A 10- to 12-fold increase in luciferase activity was observed in the cells infected with the adenovirus containing hPNPaseold-35 compared to the control cells. This activation was inhibited by compounds that reduced ROS. The authors suggest that hPNPaseold-35 plays an important role in producing age-related inflammation.
(0003643) |
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Products: Luciferase Assay System | pGL3-Control Vector | pSV-β-Galactosidase Control Vector |
| 9. |
Yamaguchi, K., Lee, S.H., Eling, T.E., and Baek, S.J.
(2004)
Identification of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) as a novel downstream target of phosphatidylinositol 3-kinase/AKT/GSK-3 pathway.
J. Biol. Chem.
279
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49617-49623
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Notes:
The LY294002 phosphatidylinositol 3-kinase (PI3K) inhibitor was used to demonstrate Nonsteroidal Anti-inflammatory Drug-activated Gene (NAG-1) as a novel downstream target of the PI3K pathway during cell activation. For these experiments, HCT-116 cells were treated with 50μM LY294002 and NAG-1 protein expression was assessed by Western blotting. Gene upregulation during LY294002 treatment was measured with a luciferase reporter construct containing the NAG-1 promoter, the pRL-null Vector as a transfection control, and the Dual-Luciferase® Assay System.
(0003262) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | LY 294002 | pRL-null Vector |
| 10. |
Chanda, S.K., White, S., Orth, A.P., Reisdorph, R., Miraglia, L., Thomas, R.S., DeJesus, P., Mason, D.E., Huang, Q., Vega, R., Yu, D., Nelson, C.G., Smith, B.M., Terry, R., Linford, A.S., Yu, Y., Chirn, G., Song, C., Labow, M.A., Cohen, D., King, F.J., Peters, E.C., Schultz, P.G., Vogt, P.K., Hogenesch, J.B., and Caldwell, J.S.
(2003)
Genome-scale functional profiling of the mammalian AP-1 signaling pathway.
Proc. Natl. Acad. Sci. USA
100
,
12153-12158
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Notes:
A human cDNA library of ~20,000 sequences was tested for putative modulators of the activator protein-1 (Ap-1) signal transduction pathway. The plasmid library was co-transfected with luciferase reporter plasmids containing AP-1, p53 or Epo response elements. Transfections were performed in 384-well plates using HEK293, HCT116 or HepG2 cells. After 48 hours, the Bright-Glo™ Luciferase Assay was used to determine the level of luciferase activity in the transfections. Luminescence was read using an Acquest Plate Reader (LJL Biosystems)
(0003299) |
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Products: Bright-Glo™ Luciferase Assay System |
| 11. |
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)
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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 |
| 12. |
Docagne, F., Nicole, O., Gabriel, C., Fernández-Monreal, M., Lesné, S., Ali, C., Plawinski, L., Carmeliet, P., MacKenzie, E.T., Buisson, A., Vivien, D.
(2002)
Smad3-dependent induction of plasminogen activator inhibitor-1 in astrocytes mediates neuroprotective activity of transforming growth factor-β1 against NMDA-induced necrosis
Mol. Cell. Neurosci.
21
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634-644
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Notes:
PAI-1 is an inhibitor of tissue-type plasminogen activator (t-PA) and has been shown to have neuroprotective activity through the TGF-β pathway. The authors performed RTPCR experiments to study PAI-1 expression in cultured neurons and astrocytes. To confirm specificity of PAI-1 amplified products, the products were cloned into pGEMT vectors and sequenced. Wildtype and PAI-1 deficient astrocytes were transfected (using the cationic lipid reagent, Transfast Transfection Reagent) with a luciferase reporter gene driven by the TGF-β1 response element (CAGA-luc) to determine if PAI-1-/- cells could transduce TGF-β1 signal. Luciferase activity was quantitated using the Luciferase Assay Kit.
(0002608) |
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Products: pGEM®-T Vector System I |
| 13. |
Hacker, H., Mischak, H., Hacker, G., Eser, S., Prenzel, N., Ullrich, A., Wagner, H.
(1999)
Cell type-specific activation of mitogen-activated protein kinases by CpG-DNA controls interleukin-12 release from antigen-presenting cells.
EMBO J.
18
,
6973-6982
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Notes:
Treatment of RAW 264.7 cells were treated with CpG-oligonucleotides derived from bacterial DNA. The cells responded by releasing TNFα and IL-12. The MEK Inhibitor U0126 inhibited the release of the proteins. The inhibitor has no effect on the TNFα promoter or mRNA levels but has a big effect on IL-12 promoter activity and mRNA levels, suggesting different mechanisms of MAPK pathway involvement. Promoter studies were performed with constructs made in the pGL3-Basic Vector and analyzed with the Luciferase Assay System.
(0001091) |
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Products: Luciferase Assay System | Luciferase Assay System with Reporter Lysis Buffer | MEK Inhibitor U0126 | pGL3-Basic Vector |
| 14. |
Agati, J.M., Yeagley, D. and Quinn, P.G.
(1998)
Assessment of the roles of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase B and protein kinase C in insulin inhibition of cAMP-induced phosphoenolpyruvate carboxykinase gene transcription.
J. Biol. Chem.
273
,
18751-18759
.
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Notes:
Up to four plasmids were transfected into H4IIe cells including 20µg of luciferase reporter vector, 2µg of pRL-SV Vector and an RSV-driven expression vector expressing the catalytic domain of PKA or ras pathway mutants. Luciferase activity was normalized to Renilla luciferase activity using the Dual-Luciferase® Reporter Assay System.
(0002063) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pRL-SV40 Vector |