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J. Biol. Chem. 286, 4644–4658. Developmentally regulated ceramide synthase 6 increases mitochondrial Ca2+ loading capacity and promotes apoptosis. 2011

Novgorodov, S.A., Chudakova, D.A., Wheeler, B.W., Bielawski, J., Kindy, M.S., Obeid, L.M. and Gudz, T.I.

Notes: The authors sought to determine the function of ceramide and ceramide synthase (CerS6) in mitochondria during post-natal brain development. They derived oligodendrocytes (OLs) from cultured, dissociated rat neonatal cortices. To determine if CerS6 plays in cell death, OLs were treated with glutamate to induce excitotoxicity, and cell death was assessed using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. To determine if the glutamate-induced excitotoxicity is dependent on ceramide or CerS6, OLs were treated with glumate in the presence or absence of the CerS6 inhibitor, fumonisin B1. To assess the mechanism of cell death resulting from ceramide-dependent glutamate toxicity in OLs, caspase activation during glutamate treatment was measured using the Apo-ONE® Homogeneous Caspase-3/7 Assay. (4171)

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Invest. Ophthalmol. Vis. Sci. 52, 890–903. Differential effects of PPARγ ligands on oxidative stress-induced death of retinal pigmented epithelial cells. 2011

Rodrigues, G.A., Maurier-Mabé, F., Shurland, D-L, Mclaughlin, A., Luhrs, K., Throo, E., Delalonde-Delaunay, L., Palleres, D., Schwighoffer, F. and Donello, J.

Notes: Serum-starved ARPE cells were treated with the oxidant, t-butylhydroperoxide (tBH), in increasing concentrations for 30 minutes, 1, 2 and 24 hours, and caspase-3/7 activity was measured using the Apo-ONE® Homogeneous Caspase 3/7 Assay to determine the mechanism of cell death. A dose-dependent increase in caspase-3/7 activity was observed. (4172)

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Current Chemical Genomics 3, 33-41. In vitro viability and cytotoxicity testing and same-well multi-parametric combinations for high-throughput screening 2009

Niles, A.L., Moravec, R.A. and Riss, T.L.

Notes: The authors review the use of in vitro cytotoxicity testing in drug discovery to characterize the toxic potential of new chemical entities (nce) at the earliest stages of profiling. DOI: 10.2174/1875397300903010033 (4000)

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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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Br. J. Cancer 92, 1430–1441.. HGS-ETR1, a fully human TRAIL-receptor 1 monoclonal antibody, induces cell death in multiple tumor types in vitro and in vivo. 2005

Pukac, L., Kanakaraj, P., Humphreys, R., Alderson, R., Bloom, M., Sung, C., Riccobene, T., Johnson, R., Fiscella, M., Mahoney, A., Carrell, J., Boyd, E., Yao, X.T., Zhang, L., Zhong, L., von Kerczek, A., Shepard, L., Vaughan, T., Edwards, B., Dobson, C., Salcedo, T. and Albert, V.

Notes: The authors examine and characterize HGS-ETR1 (Mapatumumab), a fully human agonistic monoclonal antibody with high affinity and specificity for TRAIL-R1. HGS-ETR1 induced cell death in tumor cell lines was mediated by the activation of the extrinsic and intrinsic death signaling pathways. The tumor cell lines Colo205, HCT116, H460, H2122, ST486, SW480, RL95-2, WU.86.86, ES2, A498, WM793, SNU398, JURL-MKI and TTn were plated at 10,000 cells/well in 96-well white, opaque plates. The tumor cells were treated with the HGS-ETR1 or a isotope control antibody (ICmAB). After 48 hours, cell viability was determined by the CellTiter-Glo® Luminescent Cell Viability Assay. Luminescence was detected using a Northstar luminescent plate reader. Caspase 3/7 activity was assessed in the HGS-ETR1 and ICmAB treated tumor cells using the Apo-ONE™ Homogeneous Caspase-3/7 Assay. The cells were plated at 10,000 cells/well into black-walled 96-well plates and treated with the HGS-ETR1 and ICmAB for 6 hours at 37°C. Caspase activity was measured by reading the plates at 405nm using a fluorometric plate reader. (3314)

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J. Biol. Chem. 279(18), 18353-18360. Argininosuccinate synthase expression is required to maintain nitric oxide production and cell viability in aortic endothelial cells. 2004

Goodwin, B.L., Solomonson, L.P. and Eichler, D.C.

Notes: In this study, the effect of arginosuccinate synthase (AS) on nitric oxide (NO) signaling was explored in bovine aortic endothelial cells. Expression of arginosuccinate synthase was knocked down using in-vitro-transcribed siRNAs. Using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay and the Apo-ONE® Homogeneous Caspase-3/7 Assay, it was found that reduction of AS leads to the induction of apoptosis. Detection was performed on a BMG FLUOstar spectrofluorometer. Supplying the cells with an NO donor decreased apoptosis induction. The regulatory role NO has on apoptosis is not known. (3065)

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Hum. Mol. Genet. 13(20), 2409-20. Correction of aberrant FGFR1 alternative RNA splicing through targeting of intronic regulatory elements 2004

Bruno, I.G., Jin, W., Cote, G.J.

Notes: Human U251 glioblastoma cell lines treated with antisense morpholino oligonucleotides were assessed for viability and apoptosis by multiplexing the CellTiter-Blue® Cell Viability and Apo-ONE® Homogeneous Caspase-3/7 Assays on single cell cultures. Cell viability was measured 4 hours after the addition of the CellTiter-Blue® Cell Viability Reagent to the cultures. Next, apoptosis measurements were performed on the same cell cultures by adding Apo-ONE® Homogeneous Caspase-3/7 Assay reagent to the cultures. Caspase-3/7 activity was then measured 12 hours later. (3168)

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Blood 102, 2605-2614. Cleavage of Bax to p18 Bax accelerates stress-induced apoptosis, and a cathepsin-like protease may rapidly degrade p18 Bax. 2003

Cao, X., Deng, X. and May, W.S.

Notes: The Apo-ONE® Homogeneous Caspase-3/7 Assay was used to assay apoptosis in transiently transfected EcR-293 cells with inducible expression of WT, Asp33Ala, p18, or Glu6Ala Bax.  Caspase activity was assessed at 0, 12, 24, and 36 hours post-induction for each protein in the presence or absence of ALLM, an inhibitor of both calpain and cathepsin. The researchers also measured caspase activity with the Apo-ONE® Homogeneous Caspase-3/7 Assay on A-549, K-562, and U-937 cells in the presence of calpain and cathepsin inhibitors.  (2783)

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J. Biol. Chem. 278, 19352-19357. COX2 activity promotes organic osmolyte accumulation and adaptation of renal medullary interstitial cells to hypertonic stress. 2003

Moeckel G.W., Zhang L., Fogo A.B., Hao C.M., Pozzi A. and Breyer M.D.

Notes: In this paper, caspase-3 activity was measured in mouse renal medullary interstitial cells (MMICs) using the Apo-ONE® Homogeneous Caspase-3/7 Assay. MMICs grown to confluency in 96-well plates were preincubated with 1mM betaine, sorbitol, or inositol before incubation in 600m OsM media for 24 hours. Researchers then added 100μl of Apo-ONE® Homogeneous Caspase-3/7 Assay Reagent and the plates were shaken at 300rpm for 30 seconds. The plates were then incubated for another hour at room temperature before results were read. Data is presented as the mean of 4 experiments at the emission OD538. (2673)

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Biochem. Biophys. Res. Commun. 308, 990-994. Erythropoietin protects cardiac myocytes from hypoxia-induced apoptosis through an Akt-dependent pathway 2003

Tramontano, A.F., Muniyappa, R., Black, A.D., Blendea, M.C., Cohen, I., Deng, L., Sowers, J.R., Cutaia, M.V., and El-Sherif, N.

Notes: The Apo-ONE® Homogeneous Caspase-3/7 Assay was used to demonstrate that 10U/ml of erythropoietin (EPO) can decrease apoptosis in primary cultured neonatal rat ventricular myocytes (NRVMs) under hypoxic conditions. The authors also describe using 1μM staurosporine and Ac-DEVD-CHO, at 5μl/well, as positive and negative controls, respectively. Cells were plated in 96-well tissue culture plates and treated in normoxic or hypoxic conditions for 24 hours before reading results using a Spectra Max GeminiXS fluorometer (Molecular Devices). (2839)

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Assay Drug Dev. Technol. 3, 469-477. HTS Compatible Assay for Antioxidative Agents Using Primary Cultured Hepatocytes 2003

Gaunitz, F. and Heise, K.

Notes: Primary hepatocytes from Sprauge-Dawley rats were incubated with various concentrations of tert-butylhydroperoxide to induce oxidative stress and then cell viability, cytotoxicity and apoptosis were assayed with Promega's CellTiter 96 AQueous One Solution Cell Proliferation Assay, CellTiter-Blue Cell Viability Assay, CellTiter-Glo Luminescent Cell Viability Assay, CytoTox-ONE Homogeneous Membrane Integrity Assay, Apo-ONE Homogeneous Caspase-3/7 Assay and assay systems. (2969)

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Circ. Res. 93, 664-673. Hypoxia-inducible factor-1 mediates activation of cultured vascular endothelial cells by inducing multiple angiogenic factors. 2003

Yamakawa, M., Liu, L.X., Date, T., Belanger, A.J., Vincent, K.A., Akita, G.Y., Kuriyama, T., Cheng, S.H., Gregory, R.J. and Jiang, C.

Notes: The Apo-ONE® Homogeneous Caspase-3/7 Assay was used to assess caspase activity in human pulmonary artery endothelial cells (HPAECs) treated with varying amounts of recombinant angiopoietin-4 (Ang-4) with or without the Angiopoietin-1 inhibitor, Tie-2Fc.  Results were expressed as a percent of a control.  Examination of DNA fragmentation in apoptotic nuclei in cells treated under similar conditions confirmed the results.    (2782)

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FASEB J. 17, 1364-1366. Oxygen-regulated expression of the Wilms' tumor suppressor Wt1 involves hypoxia-inducible factor-1 (HIF-1). 2003

Wagner, K.D., Wagner, N., Wellmann, S., Schley, G., Bondke, A., Theres, H. and Scholz H.

Notes: Researchers used the Apo-ONE® Homogeneous Caspase-3/7 Assay on rat kidney homogenates from normoxia (20% O2) and hypoxia (8% O2) treated rats. The authors homogenized rat kidneys in a well described buffer before clearing the homogenates by centrifugation at 50,000 x g and using them in the Apo-ONE® Homogeneous Caspase-3/7 Assay. A Genius SpectraFluorplus fluorescence spectrometer with a 521nm filter was used to measure the relative fluorescence units obtained from the kidney lysates. (2698)

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