A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers

Niles, Andrew L.; Moravec, Richard A.; Hesselberth, P. Eric; Scurria, Michael A.; Daily, William J.; Riss, Terry

doi:10.1016/j.ab.2007.04.007

Cited by 146 publications

(94 citation statements)

References 17 publications

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“…Furthermore, live and dead cell measures are normally inversely related, but here, there was a concomitant decrease, not increase, in the cytotoxic response for negative control siRNA versus transfection control. The fact that there was a reduction in both readouts indicates the possibility that colour quenching of the fluorimetric readout (Niles et al, 2007) might have occurred under conditions of negative control siRNA transfection. Alternatively, an added cytotoxic effect by targeting a survival gene could produce a similar profile, as seen in Supporting Information Fig.…”

Section: Resultsmentioning

confidence: 99%

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase‐induced cardiomyopathy: involvement of mitofusin‐2

McLaughlin

Zhao

O’Neill

et al. 2017

British J Pharmacology

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Background and PurposeThe anthracycline doxorubicin (DOX), although successful as a first‐line cancer treatment, induces cardiotoxicity linked with increased production of myocardial ROS, with Nox2 NADPH oxidase‐derived superoxide reported to play a key role. The aim of this study was to identify novel mechanisms underlying development of cardiac remodelling/dysfunction further to DOX‐stimulated Nox2 activation.Experimental ApproachNox2−/− and wild‐type (WT) littermate mice were administered DOX (12 mg·kg−1 over 3 weeks) prior to study at 4 weeks. Detailed mechanisms were investigated in murine HL‐1 cardiomyocytes, employing a robust model of oxidative stress, gene silencing and pharmacological tools.Key ResultsDOX‐induced cardiac dysfunction, cardiomyocyte remodelling, superoxide production and apoptosis in WT mice were attenuated in Nox2−/− mice. Transcriptional analysis of left ventricular tissue identified 152 differentially regulated genes (using adjusted P < 0.1) in DOX‐treated Nox2−/− versus WT mice, and network analysis highlighted ‘Cell death and survival’ as the biological function most significant to the dataset. The mitochondrial membrane protein, mitofusin‐2 (Mfn2), appeared as a strong candidate, with increased expression (1.5‐fold), confirmed by qPCR (1.3‐fold), matching clear published evidence of promotion of cardiomyocyte cell death. In HL‐1 cardiomyocytes, targeted siRNA knockdown of Nox2 decreased Mfn2 protein expression, but not vice versa. While inhibition of Nox2 activity along with DOX treatment attenuated its apoptotic and cytotoxic effects, reduced apoptosis after Mfn2 silencing reflected a sustained cytotoxic response and reduced cell viability.Conclusions and ImplicationsDOX‐induced and Nox2‐mediated up‐regulation of Mfn2, rather than contributing to cardiomyocyte dysfunction through apoptotic pathways, appears to promote a protective mechanism.Linked ArticlesThis article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc

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Section: Resultsmentioning

confidence: 99%

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase‐induced cardiomyopathy: involvement of mitofusin‐2

McLaughlin

Zhao

O’Neill

et al. 2017

British J Pharmacology

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“…At 4 h postinfection, the media were replaced again with fresh compound-containing media, and 72 h after infection the cells were assayed for firefly luciferase activity. Cell viability was monitored in parallel with an ATP assay (19). As shown in Fig.…”

Section: Identification Of Wea-derived Compounds With Antiviral Activmentioning

confidence: 99%

Pan-genotypic Hepatitis C Virus Inhibition by Natural Products Derived from the Wild Egyptian Artichoke

Elsebai

Koutsoudakis

Saludes

et al. 2016

J Virol

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Hepatitis C virus (HCV) infection is the leading cause of chronic liver diseases. Water extracts of the leaves of the wild Egyptian artichoke (WEA) [Cynara cardunculus L. var. sylvestris (Lam.) Fiori] have been used for centuries in the Sinai Peninsula to treat hepatitis symptoms. Here we isolated and characterized six compounds from the water extracts of WEA and evaluated their HCV inhibition capacities in vitro. Importantly, two of these compounds, grosheimol and cynaropicrin, inhibited HCV with half-maximal effective concentrations (EC 50 s) in the low micromolar range. They inhibited HCV entry into target cells and were active against both cell-free infection as well as cell-cell transmission. Furthermore, the antiviral activity of both compounds was pan-genotypic as HCV genotypes 1a, 1b, 2b, 3a, 4a, 5a, 6a, and 7a were inhibited. Thus, grosheimol and cynaropicrin are promising candidates for the development of new pan-genotypic entry inhibitors of HCV infection. IMPORTANCEBecause there is no preventive HCV vaccine available today, the discovery of novel anti-HCV cell entry inhibitors could help develop preventive measures against infection. The present study describes two compounds isolated from the wild Egyptian artichoke (WEA) with respect to their structural elucidation, absolute configuration, and quantitative determination. Importantly, both compounds inhibited HCV infection in vitro. The first compound was an unknown molecule, and it was designated "grosheimol," while the second compound is the known molecule cynaropicrin. Both compounds belong to the group of sesquiterpene lactones. The mode of action of these compounds occurred during the early steps of the HCV life cycle, including cell-free and cell-cell infection inhibition. These natural compounds present promising candidates for further development into anti-HCV therapeutics. Hepatitis C virus (HCV) is an enveloped, positive-strand RNA virus classified as a separate genus (Hepacivirus) within the Flaviviridae family. It shows a high degree of genetic diversity, with 7 major circulating genotypes (1). HCV is mainly transmitted through exposure to HCV-contaminated blood. Most infections remain persistent, summing up to an estimated 150 million chronic HCV carriers worldwide (2). As persistent HCV infection frequently causes chronic hepatitis that can progress to liver cirrhosis and liver cell carcinoma, it is a major threat to human health (3, 4).Treatment options for chronically infected individuals have dramatically improved over the last few years. This has been due to the development of highly potent direct-acting antivirals (DAAs) that increased sustained response rates to over 90%, even in interferon-free combinations (5). Currently approved DAAs include NS3/4A protease inhibitors (telaprevir, boceprevir, and simeprevir), NS5A inhibitors (daclatasvir and ledipasvir), and the NS5B polymerase inhibitor sofosbuvir. Further antiviral drugs are in clinical trials and are about to be approved. Nonetheless, the rapid replication of HCV, al...

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“…EREB2-5 cells were resuspended in fresh medium at 10 4 cells per 100 l and plated in duplicate in a black 96-well plate. Cytotoxicity was quantitated by using a MultiTox-Fluor multiplex cytotoxicity assay (Promega), as recommended by the manufacturer (58). Dead cells were detected by fluorescence at 485-nm excitation and 520-nm emission by using a SpectraMax plate reader (Molecular Devices).…”

Section: Methodsmentioning

confidence: 99%

“…To confirm these data, we utilized a second assay for cell viability that measures protease activity released from dead cells by cleaving a cell-impermeable fluorogenic peptide substrate (58). The dead cell protease activity from EREB2-5 cells grown in the absence of ␤-estradiol for 7 days was set to 100%.…”

Section: Ebna-2 Is Required For Mitochondrial Function In Ebv-mentioning

confidence: 99%

Convergence of Kaposi's Sarcoma-Associated Herpesvirus Reactivation with Epstein-Barr Virus Latency and Cellular Growth Mediated by the Notch Signaling Pathway in Coinfected Cells

Spadavecchia

González‐López

Carroll

et al. 2010

J Virol

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Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of primary effusion lymphoma (PEL).Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). PEL is a body cavity based lymphoma that is rapidly fatal (15,24,37,56,63). Multiple, continuous PEL cell lines were established by culturing clinical samples from PEL patients (13,54,60). These cell lines were the first tissue culture models for KSHV infection (53,60). Approximately 70% of PEL cell lines are coinfected with KSHV and EpsteinBarr virus (EBV) (17).The KSHV lytic switch protein, replication and transcriptional activator (Rta), encoded by open reading frame 50 (ORF50), is both necessary and sufficient for viral reactivation in PEL cells (47,48,65,70). Lytic reactivation requires the formation of a ternary complex between Rta, delayed early promoter DNA, and the host cell's recombination signal binding protein (RBP)-Jk (also known as CSL-1 and CBF1) (11,44,45). RBP-Jk is a sequence-specific DNA-binding protein that is the nuclear effector of the canonical Notch signal transduction pathway (23).Whereas RBP-Jk is required for productive KSHV reactivation (45), it is also required for latent (nonproductive) transformation of primary B cells by EBV (39). In this program, termed latency III, EBV nuclear antigen 2 (EBNA-2) transactivates two EBV promoters by interacting with RBP-Jk. The two promoters express transcripts that encode seven proteins that promote viral persistence by stabilizing the EBV genome, stimulating B-cell growth and expansion, and blocking B-cell apoptosis (39). One of the transforming EBV latency proteins is latent membrane protein 1 (LMP-1). LMP-1 is a constitutively active ortholog of the cellular tumor necrosis factor (TNF) receptor CD40 (55, 68). LMP-1 induces cell proliferation and transformation by engaging multiple signaling pathways including NF-B, TNF receptor-associated factors (TRAFs) 1 to 3, Akt kinase, Jun kinase, c-Rel, and p38 (16, 18-20, 27, 31, 40, 43, 46, 51, 52, 55). EBV transformation also requires transactivation of cellular genes by EBNA-2 in an RBP-Jk-dependent fashion (22,34,38,62,64,71).RBP-Jk's principal role in KSHV and EBV infection is to specify transcriptional targets of Rta and EBNA-2. RBP-Jk also specifies transcriptional targets for the activated form of the cellular Notch receptor (Notch intracellular domain 1 [NICD-1]); despite the apparent mechanistic similarity of NICD-1 transactivation to that of Rta and EBNA-2, these proteins are not always phenotypically interchangeable. For example, NICD-1 and EBNA-2 do not productively reactivate KSHV from latency (11,14,45), and NICD-1 does not fully complement EBNA-2 deficiency in long-term outgrowth of lymphoblastoid cell lines (LCLs) (25,28). Furthermore, the KSHV genome contains 177 RBP-Jk sites and yet, in the absence of de novo protein expression, Rta only transactivates eight KSHV genes * Corresponding author. Mailing address: ICPH E350C, 225 Warren St.,

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A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers

Cited by 146 publications

References 17 publications

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase‐induced cardiomyopathy: involvement of mitofusin‐2

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase‐induced cardiomyopathy: involvement of mitofusin‐2

Pan-genotypic Hepatitis C Virus Inhibition by Natural Products Derived from the Wild Egyptian Artichoke

Convergence of Kaposi's Sarcoma-Associated Herpesvirus Reactivation with Epstein-Barr Virus Latency and Cellular Growth Mediated by the Notch Signaling Pathway in Coinfected Cells

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