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Cell Chemical Biology 25, 1337–1349. APD-containing cyclolipodepsipeptides target mitochondrial function in hypoxic cancer cells. 2018

Jacobsen, K.M., Villadsen, N.L., Tørring, T., Nielsen, C.B., Salomón, T., Nielsen, M.M., Tsakos, M., Sibbersen, C., Scavenius, C., Nielsen, R., Christensen, E.I., Guerra, P.F., Bross, P., Pedersen, J.S., Enghild, J.J., Johannsen, M., Frøkiær, J., Overgaard, J., Horsman, M.R., Busk, M. and Poulsen, T.B.

Notes: Hypoxic cancer cells constitute a major challenge in chemo- and radiotherapy. The researchers present evaluation of APD-CLD compounds that induce selective cell death in hypoxic cancer cells by impeding mitochondrial function. The RealTime-Glo™ Annexin V Apoptosis Assay, CellTiter-Blue® Cell Viability Assay, CellTox™ Green Real-Time Cytotoxicity Assay and GSH-Glo™ Glutathione Assay were used to assess mechanism of action. (5168)

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Toxicology in Vitro 51, 83-94. High-throughput toxicity testing of chemicals and mixtures in organotypic multi-cellular cultures of primary human hepatic cells. 2018

Orbach, S.M., Ehrich, M.F., and Rajagopalan, P.

Notes: These authors describe the design, assembly and toxicant response of multi-cellular 3D hepatic organotypic culture models. In high-throughput screening, changes in cell viability were determined over 24 hours using the RealTime-Glo™ MT Cell Viability Assay. Mechanism of cell death was determined with the ApoTox-Glo™ Triplex Assay, and GSH concentration was measured using the GSH-Glo™ Glutathione Assay.  (5011)

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Cell 168, 210–23. Nuclear localization of mitochondrial TCA cycle enzymes as a critical step in mammalian zygotic genome activation. 2017

Nagaraj, R., Sharpley, M.S., Chi, F., Braas, D., Zhou, Y., Kim, R., Clark, A.T. and Banerjee, U. 

Notes: The authors of this study explore the hypothesis that mitochondria pass glycolytic enzymes to the nucleus during early development to assist with producing substrates needed for epigenetic changes needed for activating the zygotic genome. Embryo extracts were analyzed with the ENLITEN® ATP Assay and the ADP-Glo™ Assay for ATP:ADP ratio; glutathione levels monitored with GSH-Glo™ Assay.  Extracts were also made as directed in the NAD/NADH-Glo™ Assay manual to determine NAD and NADH levels. (4831)

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Blood 113, 744–754. CYP1B1 expression promotes the proangiogenic phenotype of endothelium through decreased intracellular oxidative stress and thrombospondin-2 expression. 2009

Tang, Y., Scheef, E.A., Wang, S., Sorenson, C.M., Marcus, C.B., Jefcoate, C.R. and Sheibani, N.

Notes: The authors tested if CYP1B1 removed cellular oxygenation products that induce oxidative stress and promote the release of antiangiogenic factors. The P450-Glo™ CYP1B1 Assay was used to determine CYP1B1 activity. The presence of glutathione was assessed using either 104 retinal endothelial cells or 50µl of mouse retinal extracts dispensed into each well of a 96-well plate with the GSH-Glo™ Glutathione Assay. (4010)

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Arterioscler. Thromb. Vasc. Biol. Sept 24, [Epub ahead of print]. Eotaxin increases monolayer permeability of human coronary artery endothelial cells. 2009

Jamaluddin, M.S., Wang, X., Wang, H., Rafael, C., Yao, Q. and Chen, C.

Notes: Glutathione levels were assessed in human coronary artery endothelial cells (HCAECs) as a measure of oxidative stress. HCAECs were treated with either 100ng/ml eotaxin, a newly discovered chemokine, or pretreated with 2µmol/l MnTBAP for 30 minutes followed by eotaxin treatment for 45 minutes. Positive controls were treatment with 10µg/ml antimycin A and 2ng/ml TNF-α. Cellular glutathione was measured using the GSH-Glo™ Glutathione Assay. (4011)

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Toxicol. Sci. 108, 35–47. 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. 2009

Reisman, S.A,, Yeager, R.L., Yamamoto, M. and Klaassen, C.D.

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. (4012)

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Expert Opin. Drug Metab. Toxicol. 4, 103–120. Bioluminescent assays for ADMET 2008

Cali, J.J., Niles, A., Valley, M.P., O’Brien, M.A., Riss, T.L., and Shultz, J.

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. (3926)

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