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Sci. Rep. 8(1), 10122. Glucose starvation induces LKB1-AMPK-mediated MMP-9 expression in cancer cells. 2018

Endo, H., Owada, S., Inagaki, Y., Shida, Y. and Tatemichi, M.

Notes: The authors investigated the role of the liver kinase B1 (LKB1)-adenosine monophosphate-activated kinase (AMPK) signaling pathway in cancer cell survival and metabolic adaptation. The CellTiter-Glo® 2.0, ROS-Glo™ and CellTiter® 96 AQueous One Solution Assays were used to monitor ATP content, reactive oxygen species and cell proliferation in LKB1 and AMPK knockdown cells. Additionally, the pGL3 Vector and Dual-Luciferase® Assay System were used to monitor matrix metalloproteinase-9 (MMP-9) promoter activity. (5162)

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Exp. Cell Res. 366(2), 181–91. HIF-dependent and reversible nucleosome disassembly in hypoxia-inducible gene promoters. 2018

Suzuki, N., Vojnovic, N., Lee, K.L., Yang, H., Gradin, K. and Poellinger, L.

Notes: The pGL3-Basic Vector was used to track activity of the PGK1 and BNIP3 gene promoter regions, and luminescence was measured using a GloMax® microplate reader. (5084)

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Gene 641, 151–60. Individual effects of the copia and gypsy enhancer and insulator on chromatin marks, eRNA synthesis, and binding of insulator proteins in transfected genetic constructs 2018

Fedoseeva, D.M., Kretova, O.V., Gorbacheva, M.A., Tchurikov, N.A.

Notes: The function of enhancer and insulator elements for the mobile elements copia and gypsy are characterized. To determine the function of these elements, cells were transfected with luciferase constructs with enhancer and insulator elements using the TransFast™ Transfection Reagent. Luciferase activity was monitored with the Dual-Luciferase® Reporter Assay. Enhancers stimulated the synthesis of long enhancer RNAs (eRNAs) and histone methylation and acetylation marks. The presence of insulator sequence reduced eRNA synthesis. (5089)

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Oncogene 37(1), 39-51. Nuclear factor E2-related factor-2 has a differential impact on MCT1 and MCT4 lactate carrier expression in colonic epithelial cells: a condition favoring metabolic symbiosis between colorectal cancer and stromal cells. 2018

Diehl, K., Dinges, L.A., Helm, O., Ammar, N., Plundrich, D., Arlt, A., Röcken, C., Sebens S., and Schäfer, H.

Notes: These authors studied the role of the antioxidant transcription factor nuclear factor E2-related factor-2 (Nrf2) in adaptation to inflammatory/environmental stress in malignant colonic epithelial cells. They used the Dual-Glo® Luciferase Assay and pGL3-Basic Vector in reporter assays, and the CellTiter® 96, Caspase-Glo® 3/7 and Glucose Uptake-Glo™ Assays to investigate Nrf2 effects on cell viability and metabolism. They found that Nrf2 has an impact on the metabolism in premalignant colonic epithelial cells exposed to inflammatory M1 macrophages, an effect accompanied by growth and survival alterations.  (5014)

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Stem Cells 36, 337–348. Differences in the Activity of Endogenous Bone Morphogenetic Protein Signaling Impact on the Ability of Induced Pluripotent Stem Cells to Differentiate to Corneal Epithelial‐Like Cells 2017

Kamarudin, T.A., Bojic, S., Collin, J., Yu, M., Alharthi, S., Buck, H., Shortt, A., Armstrong, L., Figueiredo, F.C., Lako, M.

Notes: RNA was extracted from the cells collected from differentiating hESC and hiPSC at days 0, 9, and 20 using the ReliaPrep RNA Cell Miniprep System. The RNA quality was evaluated using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, MA). One microgram of extracted RNA was converted into cDNA using reverse transcription (GoScript Transcription System). Quantitative real‐time polymerase chain reaction (qRT‐PCR) was carried out using the QuantStudio 7 Flex Real‐Time PCR System (Thermo Fisher Scientific, MA) and GoTaq qPCR Master Mix. pGL3 BRE Luciferase was used for plasmid lipofection to transfect the cells in each well of a 24-well plate. Cells that were transfected with empty vector (pGL3‐Basic) or BMP reporter (pGL3 BRE Luciferase) were cotransfected with empty Renilla vector (pRL‐Null). Luciferase activities were evaluated with a Dual‐Luciferase Assay System. (4982)

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Genes Cells 12401, [Epub ahead of print]. Application of NanoLuc to monitor the intrinsic promoter activity of GRP78 using the CRISPR/Cas9 system 2016

Oh-Hashi, K., Furuta, E., Norisada, J., Amaya, F., Hirata, Y. and Kiuchi, K.

Notes: CRISPR/Cas9 editing was used to tag the GRP78 gene with NanoLuc® luciferase in order to create a reporter of the intrinsic promoter activity of this gene. The authors used a single guide RNA against the N-terminal coding region of exon1 of the human GRP78 gene and constructed a donor gene that contains the puromycin-resistant gene following the NanoLuc® gene. The NanoLuc® gene was aligned with the puromycin-resistant gene through the 2A peptide sequence and was inserted into the pGL3-based vector (NL-2a-Puro-pGL3). To generate the donor gene, G78-NL, the N-terminal coding region that includes exon 1 of human GRP78 was inserted into the NanoLuc® gene in the NL-2a-Puro-pGL3. After lysis of the cells expressing the indicated gene in 24- or 96-well plates with passive lysis buffer, the lysate was mixed with the NanoLuc® substrate, and the luciferase activity in each well was measured using a GloMax® luminometer. In some cases, the culture medium from each well was mixed with the NanoLuc® substrate, and the activity for each was measured. (4754)

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Genes Cells 21(1), 25–40. Distal regulatory element of the STAT1 gene potentially mediates positive feedback control of STAT1 expression. 2016

Yuasa, K. and Hijikata, T.

Notes: The function of the regulatory element, 5.5URR, upstream of signal transducer and activator of transcription 1 (STAT1) is investigated. The Dual-Luciferase® Reporter Assay showed a significant increase in transcription in the presence of the 5.5URR upon interferon treatment. The HaloCHIP™ System showed a physical interaction between the 5.5URR element and the STAT1 promoter, which was stimulated by interferon treatment. Together, the 5.5URR element may serve in maintaining interferon signaling in relation to STAT1. (5097)

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Biochem. Biophys. Rep. 6, 1-8. The relationship between RUVBLI (Pontin, Tip49, NMP238) and BCL6 in benign and malignant human lymphoid tissues. 2016

Baron, B.W., Baron, R.M., and Baron, J.M.

Notes: HEK 293T cells were transfected with a reporter plasmid (assembled in the pGL3 Basic Vector) with a consensus BCL6 binding site and a BCL6 expression plasmid using the ViaFect™ Transfection Reagent (about 2µg of DNA per well in 6-well plates at 50-60% confluency). Promoter activation was monitored with a luciferase assay control with a β-galactosidase control. Western blotting of transfected cells used the Anti-Rabbit IgG (Fc), Alkaline Phosphatase Conjugate and the Western Blue® Stabilized Substrate for Alkaline Phosphatase. (4657)

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Nat. Commun. 6, 8089. Synergistic activation of human pregnane X receptor by binary cocktails of pharmaceutical and environmental compounds 2015

Delfosse, V., Dendele, B., Huet, T., Grimaldi, M.,  Boulahtouf, A., Gerbal-Chaloin, S., Beucher, B., Roecklin, D., Muller, C., Rahmani, R., Cavaillès, V., Daujat-Chavanieu, M., Vivat, V., Pascussi, J-M., Balaguer, P. and Bourguet, W.

Notes: Humans are exposed to a cocktail of low-dose chemicals in the environment, through diet, and through medication. However, most studies looking at compound toxicity, look at these compounds in isolation, not as they might be encountered in the environment—in mixtures. The pregnane X receptor (PXR) is a xenoreceptor that has been identified by the US Environmental Protection Agency as a major target of environmental and dietary chemicals, and many studies have highlighted the role of nuclear receptors like PXR in transducing the deleterious effects of endocrine disrupting compounds in the environment. The authors of this study used compound screening and functional analysis to demonstrate that the combined use of an environmentally persistent organochlorine pesticide and the active component of contraceptive pills (17α-ethinylestradiol) produces synergistic effects on PXR and expression of its target gene, the cytochrome P450 gene, CYP34A. Gene expression of CYP3A4 was measured using a luciferase reporter created in a pGL3-basic backbone. Activity of the CYP3A4 protein was measured in primary human hepatocytes using the P450-Glo™ CYP3A4 Assay with Luciferin-IPA, and cell number was normalized using the CellTiter-Glo® Luminescent Cell Viability Assay. (4579)

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PLos ONE 10, (epub ahead of print) e0128683. Wnt/β-catenin signaling regulates the expression of the ammonium permease gene RHBG in human cancer cells. 2015

Merhi, A., De Mees, C., Abdo, R., Alberola, J.V. and Marini, A.M.

Notes: The putative promoter and deletion mutants of the proposed promoter of the RBHG gene were cloned into the pGL3-Basic Vector for reporter assay investigation. Reporter plasmids and the pRL-TK Control Vector were cotransfected into HepG2 cells with the ViaFect™ Transfection Reagent (transfection details not provided). Forty-eight hours post-transfection, reporter activity was measured with the Dual-Glo® Luciferase Assay System using a GloMax® Instrument. (4685)

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EMBO J. 33, 1565-1581. MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures 2014

Muraoka, N., Yamakawa, H., Miyamoto, K., Sadahiro, T., Umei, T., Isomi, M., Nakashima, H., Akiyama, M., Wada, R., Inagawa, K., Nishiyama, T., Kaneda, R., Fukuda, T., Takeda, S., Tohyama, S., Hashimoto, H., Kawamura, Y., Goshima, N., Aeba, R., Yamagishi, H., Fukuda, K. and Ieda, M.

Notes: For construction of the Snai1 30 UTR reporter, the CMV promoter was subcloned into the promoterless pGL3-Basic vector upstream of the luciferase gene. A 755-bp Snai1 30 UTR fragment containing miR-133a-binding sites was amplified by PCR and subcloned into the modified pGL3-Basic vector. The activities of firefly luciferase and renilla luciferase in the control vector were determined by the Dual-Glo® Luciferase Assay System. RNA was extracted from MEFs, GMT-, GMT/miR-133-, or GMT/miR-133/ Snai1-induced aMHC-GFP+ cells, neonatal mouse heart tissues, HCFs, GMTMM-, GMTMM/miR-133-, GMTMM/miR-133/Snai1-transduced HCFs using ReliaPrep™ RNA Cell Miniprep System for gene microarray analysis. (4737)

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Nucl. Acids Res. 40(8), 3378-3391. Identification of CTCF as a master regulator of the clustered protocadherin genes. 2012

Golan-Mashiach, M., Grunspan, M., Emmanuel, R., Gibbs-Bar, L., Dikstein, R., and Shapiro, E.

Notes: Specific neuronal connectivity is thought to be based on the expression of the protocadherins (Pcdh)--a family of membrane adhesion proteins. Each neuron expresses only a specific subset of the Pcdh genes. The authors of this paper used a bioinformatics approach to identify conserved, gene-specific regions upstream of the Pcdh genes. They showed that this specific sequence element (SSE) is involved in transcription regulation together with a conserved sequence element (CSE), and identified a potential interacting protein partner. To demonstrate promoter activity, the SSE-CSE region was cloned upstream of the luciferase gene in the pGL3 Basic Vector and its effect on luciferase expression was evaluated. The authors then isolated protein complexes that bound the SSE-CSE region and characterized the interacting proteins by mass spectrometry. The CCTC binding-factor (CTCF) was identified as a key molecule that binds and activates Pcdh promoters. As part of the study, human CTCF  and the CTCF-binding domain were expressed in the TNT® T7 Quick Coupled Transcription/Translation System. The in vitro expressed proteins were fluorescently labeled using the FluoroTect™ GreenLys System and were used in EMSA to confirm interaction with the SSE-CSE region. (4249)

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J. Biol. Chem. 286, 37196–206. 5-Aza-2'-deoxycytidine activates iron uptake and heme biosynthesis by increasing c-myc nuclear localization and binding to the e-boxes of transferrin receptor 1 (TfR1) and ferrochelatase (Fech) genes. 2011

Ning, B., Liu., G., Liu, Y., Su, X., Anderson, G.J., Zheng, X., Chang, Y., Guo, M., Liu, Y., Zhao, Y. and Nie, G.

Notes: The authors used GoTaq® DNA Polymerase to amplify cDNA generated from total RNA (RT-PCR) extracted from murine erythroid leukemia (MEL) cells and mouse erythroid burst-forming units (BFU-Es). These cells were used to study the molecular mechanism of 5-aza-2'-deoxycytidine (5-aza-CdR)-induced erythroid differentiation, a process involved in azanucleotides for treating myelodysplastic syndromes (MDS) that reduces the risk of transformation to acute myeloid leukemia (AML). Treatment of these cells with 5-aza-CdR, a hypomethylation reagent, upregulated genes responsible for heme production and iron uptake. The pGL3 basic vector and promoter were used to create plasmid constructs of different E-box regulatory sequences with a luciferase reporter. The plasmids were cotransfected with c-Myc, Max or both transcription factors into human hepatocytes (HepG2). The Dual-Luciferase® Reporter Assay System was used to identify that the –6kb E-box of the transferrin receptor 1 (TfR1) promoter was a strong enhancer for inducing TfR1 expression when c-Myc and Max formed functional complexes that bound to it. Bisulfite sequencing was performed to study methylation patterns after 5-aza-2’-CdR treatment using the pGEM-T® Easy Vector system to ligate the isolated DNA fragments for TfR1 and Fech (ferrochetalase), which were transformed into E coli. for final sequencing. (4176)

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Biochem. Biophys. Res. Commun. 408, 160-166. Epigenetic regulation of the transcription factor Foxa2 directs differential elafin expression in melanocytes and melanoma cells. 2011

 Yu, K.S., Jo, J.Y., Kim, S.J., Lee, Y., Bae, J.H., Chung, Y.H., and Koh, S.S.

Notes: These authors showed that expression of the serine protease inhibitor elafin is regulated by epigenetically controlled expression of the transcription factor Foxa2. Treatment of melanoma cells with a DNA methyltransferase inhibitor induced elafin expression, resulting in reduced proliferation and increased apoptosis. Luciferase reporter assays were used to show that Foxa2 binding was required for activation of elafin expression, and that Foxa2 binding was activated by treatment with the methyltransferase inhibitor. These assays used a pGL3-Basic Vector construct in which expression of firefly luciferase was driven by the upstream region bearing the Foxa2 binding site. The pRL-TK Vector, expressing Renilla luciferase, was used as a normalization control. The AccessQuick™ System was used for RT-PCR analysis to show that Foxa2 mRNA was barely detectable in melanoma cells.  (4345)

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J. Biol. Chem. 286, 42863–872. Interplay between Vascular Endothelial Growth Factor (VEGF) and Nuclear Factor Erythroid 2-related Factor-2 (Nrf2), Implications for Preeclampsia. 2011

Kweider, N., Fragoulis, A., Rosen, C., Pecks, U., Rath, W., Pufe, T., and Wruck, C.J.

Notes: These authors investigated the relationship between VEGF and oxidative stress related to preeclampsia. They showed that VEGF activates Nrf2 in an ERK1/2-dependent manner, protecting against oxidative stress. They first used a dual-luciferase reporter assay and a pGL3-ARE vector construct to show that VEGF activates ARE in the cytotrophic cell line BeWo. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase® reporter assay system and the GloMax®-96 microplate luminometer. The authors then showed that inactivation of the transcription factor Nrf2 by shRNA abolished this VEGF-dependent ARE activation. To determine whether Nrf2 protected BeWo cells from oxidative stress, cells were pretreated with VEGF and then exposed to H2O2 before monitoring cell viability and cytotoxicity. Cytotoxicity assays were performed using the CytoTox-Glo™ Assay. (4199)

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PLos ONE 6(7), E22438. Krüppel-Like Factor 6 Expression Changes during Trophoblast Syncytialization and Transactivates ßhCG and PSG Placental Genes. 2011

Racca A.C., Camolotto S.A., Ridano M.E., Bocco J.L., Genti-Raimondi S., and Panzetta-Dutari, G.M.

Notes: These authors studied KLF6 expression during human trophoblast cell differentiation, and its role in the regulation of genes associated with placental development and pregnancy maintenance. They used immunofluorescence microscopy, RT-qPCR and luciferase reporter assays to investigate cellular localization, mRNA expression, and transcriptional activation. Reporter assays were performed using various luciferase reporter constructs, the Dual-Luciferase® Assay, and the GloMax®-Multi Detection System. KLF6 was shown to play a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as βhCG and PSG. (4197)

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Biochem. J. 436, 387–397. The novel Nrf2-interacting factor KAP1 regulates susceptibility to oxidative stress by promoting the Nrf2-mediated cytoprotective response. 2011

Maruyama, A., Nishikawa, K., Kawatani, Y., Mimura, J., Hosoya, T., Harada, N., Yamamato, M. and Itoh, K.

Notes: These authors first used a FLAG-tagged protein (nfr2) with a HeLa Nuclear extract and captured interacting proteins via SDS-PAGE and in-gel digests of bands to identify (Krüppel-associated box)-associated protein 1 (KAP1) as a potential interacting partner. Human KAP1 was purchased as a HaloTag® CMV Flexi® Vector from Kazusa and used in a Mammalian PullDown scenario (with HaloLink™ Resin) to demonstrate interaction between the two proteins. A reporter assay was used to show that KAP1 facilitates Nrf2 transactivation in a dose-dependent manner. The authors defined the interaction sites using GST-tagged nrf2 and various forms of KAP1-HaloTag® Fusions expressed in TNT® SP6 High-Yield Wheat Germ Extract. GST-tagged proteins were expressed in E. coli and bound to glutathione-Sepharose beads. These bound proteins were mixed with the KAP1 from the cell-free expression system, incubated for 4 hours at 4°C, washed and stained with the HaloTag® TMR Ligand for 30 minutes. The proteins from the pull-down assay were subjected to SDS-PAGE and the HaloTag® proteins detected by phosphorimaging and the GST proteins by Coomassie Brilliant Blue Staining. A two-hybrid system consisting of the pRL-TK Vector with a firefly luciferase reporter with Gal4 UAS, mouse Nrf-2 N-terminal domain and KAP1 was also used. The vectors were transfected into Nrf2 knockout MEFs for 4 hours then incubated for 36 hours before luciferase expression was determined using the Dual-Luciferase® Reporter Assay System. (4123)

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J. Lipid Res. 51, 2211–2222. Pioglitazone increases apolipoprotein A-I production by directly enhancing PPRE-dependent transcription in HepG2 cells. 2010

Zhang, L.H., Kamanna, V.S., Ganji, S.H., Xiong, X-M. and Kashyap, M.L.

Notes: The authors investigated the role of pioglitazone on transcriptional regulation of the apoA-I gene and looked at the biological properties of pioglitazone-induced apoA-I-containing high-density lipoprotein particles (HDL). To investigate the biological properties of the HDL particles, the authors treated THP-1 cells with conditioned medium from HepG2 cultures treated or untreated with pioglitazone and looked at adhesion to human aortic endothelial cells (HAEC). During the experiment, HAEC viability and proliferation were monitored using the CellTiter-Glo® Luminescent Cell Viability Assay. Additionally, to determine whether pioglitazone stimulates apoA-I transcription, a luciferase reporter construct was made containing the apoA-I gene promoter. Transfected cells were treated with pioglitazone, and luciferase expression was monitored using the Dual-Luciferase® Reporter Assay System. (4173)

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Nucl. Acids Res. 37, 2070–86. HMGB1 and HMGB2 proteins up-regulate cellular expression of human topoisomerase IIα. 2009

Stros, M., Polanská, E., Struncová, S. and Pospísilová, S.

Notes: The authors examined whether HMGB1 and HMGB2 proteins could affect promoter activity of the topoisomerase IIα gene. Portions of the topoisomerase IIα gene promoter were cloned into the pGL3 Basic Vector, and Saos-2 cells were cotransfected with the resulting constructs, an HMGB1- or HMGB2-expressing plasmid and the pRL-tk Vector as a control for normalization. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay. To determine whether HMGB1 and HMGB2 promoted binding of the transcription factor nuclear factor-Y (NF-Y) to the topoisomerase IIα promoter, the authors used a chromatin immunoprecipitation (ChIP) assay. Two populations of Saos-2 cells, one of which expressed HMGB1 or HMGB2 and one that had expression inhibited, were fixed with formaldehyde, then treated to shear chromatin. Immunoprecipitation was performed using an anti-NF-Y antibody, and the amount of DNA bound to the NF-Y was quantified by semi-quantitative PCR using GoTaq® Hot Start DNA Polymerase and Green GoTaq® Flexi Reaction Buffer. (4037)

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J. Biol. Chem. 283, 30650–7. Cyclin-dependent kinase 2 negatively regulates human pregnane X receptor-mediated CYP34A gene expression in HepG2 liver carcinoma cells. 2008

Lin, W., Wu, J., Dong, H., Bouck, D., Zeng, F.Y and Chen, T.

Notes: HepG2 cells in a T-25 culture flask (3 million cells at 70ndash;80% confluency) were transfected with CYP3A4 reporter plasmid (a pGL3 reporter construct) and a CMV-Renilla control plasmid (a total of 2.5µg of plasmid mix was used) using FuGENE® 6 Reagent for transient transfection assays. Reporter activities were measured using the Dual-Glo® Luciferase Assay System. (4268)

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Mol. Pharmacol. 73, 769-777. Dioxin-mediated up-regulation of aryl hydrocarbon receptor target genes is dependent on the calcium/calmodulin/CaMKIalpha pathway. 2008

Monteiro, P., Gilot, D., Le Ferrec, E., Rauch, C., Lagadic-Gossmann, D., and Fardel, O.

Notes: Regulation of genes targeted by the ligand-activated aryl hydrocarbon receptor (AhR) has been shown to be controlled by calcium (Ca(2+)) changes induced by AhR agonists such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This study investigated this link. As part of the study, MCF-7 cells were transfected with various pGL3 firefly luciferase reporter constructs and the control pRL-TK Vector expressing Renilla luciferase. Transfection conditions were as follows: MCF-7 cells were cultured in 24-well plates and transfection was performed using FuGENE® 6 transfection reagentwith a FuGENE:DNA ration of 3:1. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. (4362)

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Mol. Biosyst. 4, 59-65. A general system for evaluating the efficiency of chromophore-assisted light inactivation (CALI) of proteins reveals Ru(II) tris-bipyridyl as an unusually efficient "warhead". 2008

Lee, J., Yu, P., Xiao, X. and Kodadek, T.

Notes: In this paper, researchers were looking for efficient chromophores for singlet oxygen generation used for chromophore-assisted light inactivation (CALI) of proteins. The HaloTag® protein and firefly luciferase were used to test how well the chromophores performed in crude extracts and living cells. The expression vector for an epitope-tagged Luciferase-HTP protein, 3X Flag-Luc-HTP-Myc, was constructed using firefly luciferase amplified from the pGL3-Basic Vector and HaloTag® (HTP) amplified from the pHT2 Vector. The fusion protein was tested for labeling with a HaloTag® biotin ligand by transfecting HeLa cells with 8μg of 3X Flag-Luc-HTP-Myc plasmid and 80ng of pRL-SV40 Vector. After transfection, cells were lysed with Passive Lysis Buffer and 2μl of HeLa cell lysate was diluted in 48μl of PBS + BSA and incubated for 30 minutes at room temperature with increasing concentrations of a biotin-HT ligand. The samples then were incubated with streptavidin-agarose for 30 minutes at room temperature, centrifuged and the luciferase activity of 20μl of supernatant was measured using the Dual-Luciferase® Reporter Assay System. The fusion protein was also tested using two chromophore ligands, ruthenium(II)tris-bipyridyl (Ru-HaloTag®[HT]) and fluoroscein-HT at a concentration of 100nM, and both were successful as measured by the Dual-Luciferase® Reporter Assay System. An in vivo CALI was performed by transfecting HeLa cells with 100ng of 3X Flag-HTP-Luc-Myc plasmid and 1ng of pRL-SV40 Vector for 15 hours, and treating the cells with Ru-HT or F-HT for 3 hours. The cells were then irradiated for 30 minutes, placed in the dark for 30 minutes, then the cells were lysed and analyzed with the DLR Assay. (3954)

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Molecular Pharmacology Fast Forward March 11, 2008, epub ahead of print. Kavalactones protect neural cells against amyloid β peptide-induced neurotoxicity via ERK1/2-dependent Nrf2-activation 2008

Wruck, C.J., Götz, M.E., Herdegen, T., Varoga, D., Brandenburg, L-O. and Pufe, T.

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. (3859)

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Nucl. Acids Res. 36, 5391–404. miR-16 family induces cell cycle arrest by regulating multiple cell cycle genes. 2008

Liu, Q., Fu, H., Sun, F., Zhang, H., Tie, Y., Zhu, J., Xing, R., Sun, Z. and Zheng, X.

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. (3894)

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Nucl. Acids Res. 36, 2107–2722. PDZ domain-mediated dimerization and homeodomain-directed specificity are required for high-affinity DNA binding by SATB1. 2008

Purbey, P.K., Singh, S., Kumar, P.P., Mehta, S., Ganesh, K.N., Mitra, D. and Galande, S.

Notes: To learn about the ideal target binding sequence for SATB1, the T-lineage-enriched chromatin organizer and transcription factor, random oligonucleotides underwent SELEX and five rounds of selection by EMSA. The enriched library of oligos was cloned into the pGEM®-T Easy Vector, transformed and sequenced. Several variants of SATB1-binding consensus sequences were annealed, ligated into the pGL3-Promoter Vector and cotransfected into HEK 293 cells with a plasmid that either contained SATB1 or was empty. After 48 hours, the cells were harvested and luciferase activity measured. The CheckMate™ Mammalian Two-Hybrid System was used to assess how the N-terminal PDZ domain of SATB1 interacted with the Cut and homeodomain in the C-terminus. (3982)

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