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Sci. Signal. 5, ra64. FAM123A binds to microtubules and inhibits the guanine nucleotide exchange factor ARHGEF2 to decrease actomyosin contractility 2012

Siesser, P.F., Motolese, M., Walker, M.P., Goldfarb, D., Gewain, K., Yan, F., Kulikauskas, R.M., Chien, A.J., Wordeman, L. and Major, M.B.

Notes: To better understand what roles FAM123A may play in signaling, cell behavior and human disease, HT1080 sarcoma cells were plated on MatTek dishes coated with 5mg/ml fibronectin before transfection with Venus (a yellow fluorescent protein)-tagged FAM123A or Venus-WTX, another member of the FAM123 gene family, using FuGENE® HD Transfection Reagent. The cells were imaged the next day for low-resolution analysis. For a higher magnification, dynamic analysis, HT1080 cells were plated onto Delta T dishes coated with 5mg/ml fibronectin and transfected with EGFP-FAM123A and FuGENE® HD Transfection Reagent. The next day, cell images were captured every 10 seconds. Examining the effect of silencing FAM123A, a reporter assay used the pGL4.34[luc2P/SRFRE/Hygro] Vector cotransfected with a CMV-Renilla coreporter and other effector plasmids or siRNA, and luciferase levels assessed using the Dual-Glo® Luciferase Assay System. (4246)

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PLos ONE 7(1), e30061. Inhibition of firefly luciferase by general anesthetics: effect on in vitro and in vivo bioluminescence imaging. 2012

Keyaerts, M., Remory, I., Caveliers, V., Breckpot, K., Bos, T.J., Poelaert, J., Bossuyt, A., and Lahoutte, T.

Notes: These authors investigated the effects of various anesthetics on bioluminescence imaging with firefly luciferase. They observed decreases in luminescence with volatile anaethetics, and found increased luciferase expression with injectable anaethetics in intact cells, but not in cell lysates in vitro. They concluded that the decreases in luciferase activity observed with volatile anaesthesia were due to hemodynamic effects, and not due to a direct inhibitory effect on luciferase enzyme itself. The apparent enhancement of luciferase activity with certain injectable anaesthetics appeared to be due to cytotoxic effects that resulted in increased permeablity to luciferase, as the same enhancement was not observed in cell lysates. D-Luciferin was used for in vivo imaging experiments. The pGL4.10 vector (encoding firefly luciferase), Luciferase Assay Reagent, and the GloMax® 96 Microplate Luminometer were used for in vitro assays using cell lysates. (4190)

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Cancer Res. 72, 810-820. SMYD3 Promotes Cancer Invasion by Epigenetic Upregulation of the Metalloproteinase MMP-9. 2012

Cock-Rada, A.M., Medjkane, S., Janski, N., Yousfi, N., Perichon, M., Chaussepied, M., Chluba, J., Langsley, G., and Weitzman, J.B.

Notes: These authors investigated the role of matrix metalloproteinase (MMP-9) in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. They found that gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. The H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. They therefore investigated the role of SMYD3 overexpression on MMP-9 expression and cell migration, identifying SMYD3 as an important new regulator of MMP-9 transcription. During the study they used the GloMax® Multi Luminometer to measure luminescence and absorbance in reporter and cell viability assays. They also used the Dual-Luciferase® Reporter Assay to measure SMYD3 activity in cells transfected with a SMYD3 reporter, and the pGL4-hRluc/TK plasmid for normalization of the experimental reporter activity. GoTaq® DNA polymerase was used in semi-quantitative RT-PCR. (4189)

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Nucl. Acids Res. 39(3), e16. Characterization of L1 retrotransposition with high-throughput dual-luciferase assays. 2011

Xie, Y., Rosser, J.M., Thompson, T.L., Boeke, J.D., and Wenfeng, A.

Notes: This paper describes a rapid dual-luciferase-based assay for L1 retrotransposition that is amenable to high-throughput screening. A firefly luciferase vector in which the luciferase gene was disrupted by an antisense intron was constructed by introducing a 900-bp fragment of the human γ-globin intron into pGL4.13. This Fluc gene, interrupted by an antisense intron, gives only minimal luciferase expression unless the luciferase gene is restored by a retrotransposition event. The authors also tested a similar retrotransposition reporter using the pGL4.73 Renilla luciferase vector, but found that the firefly construct gave much higher signals. They therefore used the firefly luciferase retrotransposition reporter, a Renilla luciferase normalization control and the Dual-Luciferase® Assay to characterize profiles of retrotransposition by various human and mouse L1 elements, and to measure the kinetics of L1 retrotransposition in cultured cells. The GloMax® Multi Luminometer was used to quantify luciferase activity. (4205)

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Brain 134, 678–92. Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the Nrf2 antioxidant pathway 2011

Linker, R.A., Lee, D-H., Ryan, S., van Dam, A.M., Conrad, R., Bista, P., Zeng, W., Hronowsky, X., Buko, A., Chollate, S., Ellrichmann, G., Brück, Dawson, K., Goelz, S., Wiese, S., Scannevin, R.H., Lukashev, M. and Gold, R.

Notes: The authors of this paper investigated the molecular mechanisms through which fumaric acid esters affect the progression and pathology of multiple sclerosis. The authors looked at the activation of the Nrf2 transcriptional pathway which plays a role in defense against oxidative stress. A stable reporter cell line expressing eight copies of the gluthione-S-transferase 2 antioxidative response elements cloned upstream of the luciferase complementary DNA in a pGL4.26 vector. Reporter cells were stimulated with dimethylfumarate for 24 hours and luciferase activity measured using the Luciferase Assay System. A strong dose-dependent induction of antioxidative response elements was observed. Next, the authors looked at the effects of fumaric acid esters on the inhibitor of Nrf2, Keap 1. C-terminal, V5-tagged Keap1 protein was transfected into 293 cells. Forty-eight hours post transfection, cells were treated with dimethyl fumarate. Samples were lysed, Keap1-V5 was immunoprecipitated and gel purified. The protein was digested with trypsin in the presence of ProteaseMAX™ Surfactant, Trypsin Enhancer. Peptide pools were analyzed using liquid chromatography-tandem mass spectrometry. The authors were able to show that the Keap1 protein was modified in response to treatment. (4215)

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Comb. Chem. High Throughput Screen . Two Panels of Steroid Receptor Luciferase Reporter Cell Lines for Compound Profiling 2011

Sedlák, D. Paguio, A. and Bartůněk, P.

Notes: pGL4.35  and pGL4.36 reporter vectors were used. (4264)

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Current Chemical Genomics 4, 43–9. Improved Dual-Luciferase Reporter Assays for Nuclear Receptors 2010

Paguio, P., Stecha, P., Wood, K.V. and Fan. F

Notes: pGL4.15[luc2P /Hygro] or pGL4.14 [luc2/Hygro] were used. (4265)

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J. Biol. Chem. 284, 20946–20955. B cell lymphoma (Bcl)-2 protein is the major determinant in bcl-2 adenine-uridine-rich element turnover overcoming HuR activity. 2009

Ghisolfi, L., Calastretti, A., Franzi, S., Canti, G., Donnini, M., Capaccioli, S., Nicolin, A. and Bevilacqua, A.

Notes: To examine post-transcriptional regulation of B cell lymphoma (bcl)-2 mRNA in human cell lines, the bcl-2 open reading frame was cloned into the pCI-neo Mammalian Expression Vector with a FLAG tag. This construct was transfected into U2OS human osteosarcoma cells, lysed, immunoprecipitated on Anti-FLAG-coated beads and analyzed by SDS-PAGE. A 260bp fragment containing the human c-myc 3´UTR adenine-uridine(AU)-rich element (ARE) was cloned into the pGL4.71 [hRlucP] Vector above to produce the pGL4.71PmARE plasmid. For transient expression, SK-N-BE and HEK293 cells in 96-well plates were cotransfected with 200ng of the pGL4.71 constructs (carrying c-myc or bcl-2 ARE) and 200ng of the pGL3-Control Vector. Luciferase expression was assessed using the Dual-Glo® Luciferase Reporter Assay System. HEK293 cells were also stably cotransfected with the same pGL4.71 constructs and a vector carrying G418 resistance. Renilla luciferase expression was assessed and clones chosen with similar expression levels. (4071)

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DNA Research 15, 137-149. Exploration of human ORFeome: High-throughput preparation of ORF clones and efficient characterization of their protein products. 2008

Nagase, T., Yamakawa, H., Tadokoro, S., Nakajima, D., Inoue, S., Yamaguchi, K., Itokawa, Y., Kikuno, R.F., Koga, H. and Ohara, O.

Notes: These authors used the Flexi® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi® System and Gateway® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

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J. Virol. 81, 558–567. Hijacking components of the cellular secretory pathway for replication of poliovirus RNA. 2007

Belov, G.A., Altan-Bonnet, N., Kovtunovych, G., Jackson, C.L., Lippincott-Schwartz, J. and Ehrenfeld, E.

Notes: In this study, the Renilla luciferase gene from the phRL-CMV Vector was incorporated into polioviral RNA in place of a structural protein-coding region. Renilla luciferase activity was then monitored as a convenient indicator of viral replication. Specifically, these authors tested whether the guanine nucleotide exchange factors GBF1 and BIG2 could rescue brefeldin A (BFA)-induced inhibition of polioviral replication in HeLa cells. Cells were transfected with plasmids encoding either GBF1 or BIG2, together with the firefly-luciferase-encoding pGL4.13 Vector, which was used as a control to monitor transfection efficiency. Eighteen hours post-transfection, the cells were transfected with polioviral replicon RNA containing the Renilla luciferase gene. One hour later, normal growth medium containing EnduRen® Live Cell Substrate and either BFA or solvent alone, was added and Renilla luciferase activity was monitored at hourly intervals for 7 hours. Cells were then lysed and firefly luciferase activity was measured to assess transfection efficiency using the Dual-Glo® Luciferase Assay System System. In the presence of BFA, GBF1 was able to partially rescue viral replication. In the presence of BIG2 and BFA, no viral replication occurred. (3562)

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Proc. Natl. Acad. Sci. USA 104, 66-71. Oxidized messenger RNA induces translation errors. 2007

Tanaka, M., Chock, P.B., and Stadtman, E.R.

Notes: Oxidative damage has been associated with a range of age-related neurological conditions. In this study, the effect of mRNA oxidation was investigated. A direct correlation was observed between the extent of oxidation and the frequency of translation errors. The authors excised the firefly luciferase (luc2) gene from the pGL4.14 Vector, attached a FLAG tag to the 5´ terminus and a Myc tag to the 3´ terminus, and subcloned the gene into a pGEM-4Z Vector that had been modified to append a poly(A) sequence. The construct was transfected into HEK293 cells, which were then cultured in the presence of an oxidizing agent. The occurrence of truncated protein fragments and short peptides increased in the presence of the oxidizing agent in a concentration-dependent manner. The effects of oxidation of mRNA were also investigated in in vitro translation experiments using mRNA treated with an iron-ascorbate mixture and hydrogen peroxide. Translation in vitro was performed using rabbit reticulocyte lysate supplemented with protease inhibitors. The translation products were detected using anti-FLAG and anti-c-Myc antibodies. (3630)

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Proc. Natl. Acad. Sci. USA 103, 16834-16839. A genetic variant that disrupts MET transcription is associated with autism. 2006

Campbell, D.B., Sufcliffe, J.S., Ebert, P.J., Militerni, R., Bravaccio, C., Trillo, S., Elia, M., Schneider, C., Melmed, R., Sacco, R., Persico, A.M. and Levitt, P.

Notes: In this family-based study of 1,231 autism cases, a genetic association of a common C allele in the promoter of the MET gene and autism was identified. Initial sequencing of the MET genes from 86 individuals with autism was used to identify several candidate variants in the MET promoter and 3´ untranslated region. Family-based analyses were then performed to determine whether an association could be demonstrated between any of these variants and autism. A G/C variant 20bp 5´ of the MET transcription start site was found to be overrepresented among individuals with autism, particularly among families where more than on child was affected. Once the candidate variant was identified, the effect of the G/C change on transcription of the MET gene was investigated in a reporter assay. Two 762 bp fragments of the MET promoter region, differing only in the G/C nucleotide, were cloned into pGL4.10 firefly luciferase reporter vectors. These vectors were then transfected into mouse neural cell lines, and luciferase production was monitored using the Dual-Glo™ Assay. The construct containing the C allele produced less than half of the luciferase activity of construct containing the G allele. (3579)

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Nucl. Acids Res. 34, 6640–6652. Brn-3b enhances the pro-apoptotic effects of p53 but not its induction of cell cycle arrest by cooperating in trans-activation of bax expression. 2006

Budhram-Mahadeo, V.S., Bowen, S., Lee, S., Perez-Sanchez, C., Ensor, E., Morris, P.J. and Latchman, D.S.

Notes: Previously, the POU domain of Brn-3a was shown to interact with p53 and increase cell survival. In this article, the authors explored the possibility that Brn-3b, which shares a POU domain 95% identical to Brn-3a, may interact with p53 and affect its role in apoptosis. To test the protein:protein interaction, GST-Brn-3b fusion protein was bound to glutathione Sepharose beads and incubated with 35S-methionine labeled full-length or truncated p53, prepared using the TNT® T7 rabbit reticulocyte lysate. The luciferase control included in the kit was used as the noninteracting protein control. After washing, the bound proteins were resolved by 12% SDS-PAGE, and the bands examined by radiography. To examine the effect of Brn-3b on two p53-regulated genes, Bax and p21cip1/waf1, ND7 cells were transiently transfected with empty vector, Brn-3b, Brn-3a or p53, or Brn-3 with p53. The reporter gene cotransfected was under the control of wildtype Bax, wildtype p21cip1/waf1, or mutant Bax. A control vector (Renilla luciferase driven by the thymidine kinase promoter) was used for normalization. Forty-eight hours posttransfection, the cells were harvested and reporter levels assessed using the Dual-Luciferase® Reporter Assay System. (3597)

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J. Clin. Oncol. 24, 983-7. Elevated serum B-lymphocyte stimulator levels in patients with familial lymphoproliferative disorders. 2006

Novak, A.J., Grote, D.M., Ziesmer, S.C., Kline, M.P., Manske, M.K., Slager, S., Witzig, T.E., Shanafelt, T., Call, T.G., Kay, N.E., Jelinek, D.F., Cerhan, J.R., Gross, J.A., Harder, B., Dillon, S.R. and Ansell, S.M.

Notes: To test the significance of the C or T polymorphism at position -871 of the serum B-lymphocyte stimulator (BLyS) promoter region, the BLyS promoter was amplified, digested with Kpn I and cloned into the pGL3-Enhancer Vector. HL60 cells were then transiently transfected by electroporation using 10µg of the pGL3-BLyS-promoter constructs and 40ng of the pGL4.75[hRluc/CMV] Vector, which expresses Renilla luciferase. After 48 hours, expression of the reporter genes was assessed using the Dual-Luciferase® Reporter Assay System. (3343)

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Cancer Res. 66, 11613-11622. HMGA1 is a determinant of cellular invasiveness and in vivo metastatic potential in pancreatic adenocarcinoma. 2006

Liau, S-S, Jazag, A. and Whang, E.

Notes: The HMGA1 proteins are known to play an important role in transcriptional regulation of human gene expression, and are overexpressed in several malignancies. This study investigated the role of HMGA1 in cellular invasiveness and metastasis of pancreatic adenocarcinoma. Overexpression of HMGA1 in the human pancreatic cell line MiaPaCa2 resulted in increased invasiveness in a Matrigel assay, and shRNA silencing of HMGA1 expression resulted in reduced invasiveness and reduced the activity of matrix metalloproteinase-9 (MMP-9), an important mediator of malignant cell invasiveness. The authors then used a reporter assay to investigate the effect of changes in HMGA1 expression on MMP-9 activity. Vectors either overexpressing HMGA1, expressing an shRNA directed against HMGA1, or expressing a control shRNA, were cotransfected into MiaPaCa2 cells along with a pGL4.12 firefly luciferase reporter vector construct containing the MMP-9 promoter upstream of the luciferase gene. MMP-9 promoter activity was significantly decreased upon HMGA1 silencing. The authors then showed that HMGA1 silencing resulted in decreased invasiveness and reduced tumor growth and MMP-9 activity in an in vivo model. (3578)

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Nucl. Acids Res. 34, e107. Identification of miRNA targets with stable isotope labeling by amino acids in cell culture. 2006

Vinther, J., Hedegaard, M.M., Gardner, P.P., Andersen, J.S. and Arctander, P.

Notes: These authors used stable-isotope labeling of amino acids in culture (SILAC), a method that allows quantitation of relative protein abundance between populations, to investigate the effect of the microRNA miRNA-1 on the HeLa cell proteome. HeLa cells grown in the presence of labeled arginine and lysine were transfected with miRNA-1, and the labeled proteins compared to those from control cells treated with the transfection reagent alone. A set of 16 proteins repressed by miRNA-1 was identified. The 3´ UTR's from 11 of the miRNA-1-regulated genes were tested in a reporter assay, and 6 were shown to repress expression in an miRNA-1-dependent fashion. For the reporter assays, the HSV TK promoter was amplified from the pRL-TK Vector and subcloned into the pGL4.12(luc2CP) Vector, creating the pGL4.12-TK vector. The 3´ UTR regions from suspected target genes were then amplified and subcloned into the pGL4.12-TK construct. The various pGL4.12-TK constructs (0.9µg) were then co-transfected along with pRL-TK (0.1µg) and miRNA-1 (50pmol) into HeLa cells (80,000 cells/well in 12-well plates). Twenty-two hours post-transfection, firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. (3561)

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Am. J. Hum. Genet. 78, 815-826. Serotonin transporter promoter gain-of-function genotypes are linked to obsessive-compulsive disorder. 2006

Hu, X., Z., Lipsky, R.H., Zhu, G., Akhtar, L.A., Taubman, J., Greenberg, B.D., Xu, K., Arnold, P.D., Richter, M.A., Kennedy, J.L., Murphy, D.L., and Goldman, D.

Notes: Three alleles were identified in the promoter of the serotonin transporter, HTTLPR, based on the number of a specific repeat sequence and a single nucleotide polymorphism found in the repeat sequence. Sequences from these alleles (named S, LA, and LG) were cloned into the pGL4.10 [luc2] Vector. These constructs were then tested by transfecting them into RN46A cells derived from rat dorsal raphe neurons. The constructs containing the LG and S allele sequences displayed a 2.8 fold increase in activity compared to the construct containing the LA allele sequence. Data was represented as relative luciferase levels compared to a control (pGL4.10 [luc2] Vector). (3411)

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Proc. Natl. Acad. Sci. USA 103, 10074-10079. The BMAL1 C terminus regulates the circadian transcription feedback loop. 2006

Kiyohara, Y.B., Tagao, S., Tamanini, F., Morita, A., Sugisawa, Y., Yasuda, M., Yamanaka, I., Ueda, H., van der Horst, G.T.J., Kondo, T. and Yagita, K.

Notes: The transcription factors BMAL1 and CLOCK form a heterodimer that positively regulates the Period (Per) and Cryptochrome (Cry) genes, which are involved in maintenance of circadian oscillations in mammals. The PER and CRY proteins negatively regulate the activity of the BMAL1/CLOCK heterodimer. To investigate the specific role of BMAL1 in maintenance of circadian rhythms, Per or Bmal1 promoter-driven luciferase reporter vectors were created using the pGL4.11[luc2P] luciferase reporter vector. Forty-eight hours after transfection of Rat-1 fibroblasts with each reporter construct, cellular clocks were synchronized by treatment with 100nM dexamethasone, and rhythmic expression of the luciferase reporter was observed. The effect of various mutant BMAL1 constructs on luciferase expression was then evaluated using this model system. (3472)

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