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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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Biochem. Pharmacol. October 24, epub ahead of print. Ibandronate  increases the expression of the pro-apoptotic gene FAS by epigenetic mechanisms in tumor cells. 2012

Thaler, R., Spitzer, S., Karlic, H., Berger, C., Klaushofer, K. and Varga, F.

Notes: Caspase-Glo® 3/7 and Caspase-Glo® 8 Assays were used to assess activation of apoptosis pathways in MC3T3-E1 cells (clonal mouse), U-2 OS human osteoscarcoma cell line and CCL-51 cells (mouse mammary gland tumor cells). Although ibandronate reduced cell proliferation in all cell lines, its effect on activation of caspases was different in neoplastic versus non-neoplastic cells. Caspase-8 and caspase-3/7 activities were reduced in MC3T3-E1 cells after 72 hours treatment with ibandronate. In two tumor cell lines assayed, opposite results were seen: caspase-8 and caspase-3/7 activities increased in U-2 OS cells and in CCL-51 cells. Luminescence was detected using a GloMax® 96 Microplate Luminometer. (Figure 1 in the paper)

To analyze FAS promoter methylation levels, fragments of the targeted promoter regions were generated by digestion of genomic DNA using CpG methylation insensitive restriction enzymes MboII and PstI from cells cultured in the presence of ibandronate for varying lengths of time. The authors demonstrated that FAS promoter methylation is altered in tumor cells in the presence of ibandronate. (4253)

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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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Biotechniques 51, 105–110. A bioluminescent assay for the sensitive detection of proteases. 2011

Leippe, D.M., Nguygen, D., Zhou, M., Good, T., Kirkland, T., Scurria, M., Bernad, L., Ugo, T., Vidugiriene, J., Cali, J.J., Klaubert, D. and O'Brien, M.

Notes: The ability to detect trace protease activity is important for assessing protein purification methods during process development and for confirming the absence of protease in final purified proteins. The authors describe a general protease assay using five peptide-conjugated luciferase substrates and compare it to fluorophore-conjugated general protease assays. (4156)

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J. Clin. Invest. 121(10), 3965-80. CD2AP in mouse and human podocytes controls a proteolytic program that regulates cytoskeletal structure and cellular survival. 2011

Yaddanapudi, S., Altintas, M.M., Kistler, A.D. et al.

Notes: In this study, the GloMax®-96 Microplate Luminometer was used to measure luciferase activity as a normalization control in transfection experiments. (4206)

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Blood 117(1), 352-61. IL-6 in human cytomegalovirus secretome promotes angiogenesis and survival of endothelial cells through the stimulation of survivin. 2011

Botto, S., Streblow, D.N., DeFilippis, V.,  White, L., Kreklywich, C.N., Smith, P.P., Caposio, P.

Notes: In this study, caspase-3/7 activity in HUVECs was measured using the Caspase-Glo® 3/7 Assay. Luminescence was measured with the GloMax®-96 Microplate Luminometer. (4207)

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J. Natl. Cancer Inst. 103, 645–61. Induction of MET by Ionizing Radiation and Its Role in Radioresistance and Invasive Growth of Cancer. 2011

De Bacco F., Luraghi P., Medico E., Reato G., Girolami F., Perera T., Gabriele P., Comoglio P.M., and Boccaccio C.

Notes: Human tumor cell lines were subjected to therapeutic doses of ionizing radiation, and MET mRNA, and protein expression and signal transduction were compared in treated and untreated cells. Luciferase reporter assays were used to evaluate the activity of the MET promoter in irradiated cells. The construct pGL2-3.1 encoding the luciferase gene under control of the human full-length MET promoter and a control promoterless pGL2-Basic Vector were transfected into cells prior to irradiation. For RNA interference experiments, cells were transfected with siRNAs 24 hours before the promoter plasmids were introduced. Luciferase activity was measured on a GloMax® 96-well Microplate Luminometer using the Luciferase Reporter Assay System. After irradiation, MET expression in cell lines was increased up to fivefold.   (4200)

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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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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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