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Oncogene Epub ahead of print. CPT1A-mediated fatty acid oxidation promotes colorectal cancer cell metastasis by inhibiting anoikis. 2018

Wang, Y.N., Zeng, Z.L., Lu, J., Wang, Y., Liu, Z.X., He, M.M., Zhao, Q., Wang, Z.X., Li, T., Lu, Y.X., Wu, Q.N., Yu, K., Wang, F., Pu, H.Y., Li, B., Jia, W.H., Shi, M., Xie, D., Kang, T.B., Huang, P., Ju, H.Q. and Xu, R.H.

Notes: The NADPH/NADP and GSH/GSSG ratios were measured in cells to determine that CPT1A is required to maintain ROS homeostasis. (5039)

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J. Anesth. 32, 120–31. Cytotoxicity of propofol in human induced pluripotent stem cell-derived cardiomyocytes. 2018

Kido, K., Ito, H., Yamamoto, Y., Makita, K. and Uchida, T.

Notes: The ATP levels of human iPSC-derived cardiomyocytes were measured using the Mitochondrial ToxGlo™ Assay. To assess cell viability and caspase-3/7 activity, the ApoTox-Glo™ Triplex Assay was used and luminescence measured on a GloMax® plate reader for the caspase-3/7 assay. Cardiomyocytes were lysed and NAD+ and NADH measured from the same well using the NAD/NADH-Glo™ Assay to assess any changes in the NAD+/NADH ratio. (4952)

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J. Biol. Chem. 293, 1504–14. Diverse NADase effector families mediate interbacterial antagonism via the type VI secretion system. 2018

Tang, J.Y., Bullen, N.P., Ahmad, S. and Whitney, J.C.

Notes: To determine relative NAD+ levels in bacteria, overnight cultures of E. coli strains with expression plasmids were diluted into LB broth, grown to mid-log phase and protein expression induced. After 1 hour, cultures were diluted to A600 = 0.5, 500µl of cells pelleted, lysed and treated with 0.4M HCl for 15 minutes. After neutralizing the cells, the lysate was mixed with an equal volume of NAD/NADH-Glo™ Detection Reagent and luminescence measured continuously for 2 hours. The measurements were compared to an E. coli strain with a control vector to calculate relative NAD+. (4950)

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Cell Death Differ. 25, 542–72. Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases. 2018

Connolly, N.M.C., Theurey, P., Adam-Vizi, V., Bazan, N.G., Bernardi, P., Bolaños, J.P., Culmsee, C., Dawson, V.L., Deshmukh, M., Duchen, M.R., Düssmann, H., Fiskum, G., Galindo, M.F., Hardingham, G.E., Hardwick, J.M., Jekabsons, M.B., Jonas, E.A., Jordán, J., Lipton, S.A., Manfredi, G., Mattson, M.P., McLaughlin, B., Methner, A., Murphy, A.N., Murphy, M.P., Nicholls, D.G., Polster, B.M., Pozzan, T., Rizzuto, R., Satrústegui, J., Slack, R.S., Swanson, R.A., Swerdlow, R.H., Will, Y., Ying, Z., Joselin, A., Gioran, A., Moreira Pinho, C., Watters, O., Salvucci, M., Llorente-Folch, I., Park, D.S., Bano, D., Ankarcrona, M., Pizzo, P. and Prehn, J.H.M.

Notes: This review article offers guidelines and protocols for assessing mitochondrial dysfunction and its role in human disease. The NAD/NADH-Glo™ Assay is mentioned in the section on mitochondrial NAD(P)H. (4949)

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Cell Death Dis. 9, 55. Mitochondrial glutamine metabolism via GOT2 supports pancreatic cancer growth through senescence inhibition. 2018

Yang, S., Hwang, S., Kim, M., Seo, S.B., Lee, J.H. and Jeong, S.M.

Notes: Human cultured cells were plated at 1,000 cells/well in 100µl of medium in 96-well plates. The next day, the growth medium was replaced with fresh medium or medium containing 4mM oxaloacetate or aspartate. After 3 days, cell viability was analyzed using the CellTiter-Glo® Luminescent Cell Viability Assay. To assess the ratio of NADPH to NADP+, cells were lysed, mixed with the NADP/NADPH-Glo® Assay reagents and luminescent measured after 30 minutes. (4948)

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Cancers (Basel) 10, E40. Regulation of cancer stem cell metabolism by secreted frizzled-related protein 4 (sFRP4). 2018

Deshmukh, A., Arfuso, F., Newsholme, P. and Dharmarajan, A.

Notes: Cancer stem cells (CSCs; 10,000 cells/well in 96-well plates) were washed with PBS and 50µl of 1mM 2-Deoxy-d-Glucose (2DG) added. After 90 minutes, glucose uptake was measured using the Glucose Uptake-Glo™ Assay. CSCs were isolated from cell lines and plated at 10,000 cells/well in 96-well plates. After growing in defined medium supplemented with various amounts of glucose plus glutamine and growth factors for 2 days, cells were treated for 24 hours and 2µl samples removed and added to 98µl of PBS. Extracellular metabolites were assessed using the Glutamine/Glutamate-Glo™ Assay. Isolated CSCs were plated at 10,000 cells/well in 96-well plates, grown for 3 days, treated for 24 hours, medium removed and cells lysed. The lysates were treated to preserve NAD+ and NADH before mixing with the NAD/NADH-Glo™ Detection Reagent and luminescence measured. (4959)

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J. Med. Chem. 61, 745–59. α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) inhibitors as novel modulators of de novo nicotinamide adenine dinucleotide (NAD+) biosynthesis. 2018

Pellicciari, R., Liscio, P., Giacchè, N., De Franco, F., Carotti, A., Robertson, J., Cialabrini, L., Katsyuba, E., Raffaelli, N. and Auwerx, J.

Notes: Human primary hepatocytes at a density of 2 × 104 were seeded into 96-well collagen-coated plates, incubated overnight and then stimulated with two compounds. After 48 hours, the levels of NAD+ was assessed using the NAD/NADH-Glo™ Assay. HepG2 and AML12 cells (5 × 103) were treated with test compounds for 4 hours in a 384-well plate. Cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay and cell necrosis evaluated using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (4953)

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Nat Chem. Biol. 13, 1088-95. A genetically encoded tool for manipulation of NADP+/NADPH in living cells 2017

Cracan, V., Titov, D.V., Shen, H., Grabarek, Z., Mootha, V.K.

Notes: NAD+/NADH-Glo and NADP+/NADPH-Glo were used to determine levels of NAD+, NADH, NADP+ and NADPH. (4990)

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Mol. Metab. 6, 535-47. NNT reverse mode of operation mediates glucose control of mitochondrial NADPH and glutathione redox state in mouse pancreatic β-cells 2017

Santos, L.R.B., Muller, C., de Souza, A.H., Takahashi, H.K., Spegel, P., Sweet, I.R., Chae, H., Mulder, H., Johas, J.-C.

Notes: NAD+/NADH-Glo and NADP+/NADPH-Glo were used to quantify NAD+, NADH, NADP+, and NADPH. (4992)

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Nat Chem. Biol. 13, 1088–95. A genetically encoded tool for manipulation of NADP+/NADPH in living cells. 2017

Cracan, V., Titov, D.V., Shen, H., Grabarek, Z. and Mootha, V.K.

Notes: Various types of HeLa cell lines (Tet3G luciferase, LbNOX, mitoLbNOX, TPNOX or mitoTPNOX) cells were seeded at 400,000 cells per 6cm dish in 3ml of DMEM without pyruvate. After growing for 24 hours, the medium replaced, grown another 24 hours and the medium replaced again. After 2 hours, medium was removed, cells washed with ice-cold PBS, scraped and divided into two 200µl aliquots. One tube of lysate was left untreated, the other had ascorbic acid added to prevent oxidation of NAD(P)H into NAD(P)+. After heating at 60°C for 20 minutes, the levels of NAD+, NADH, NADP+ and NADPH were determined using the NAD/NADH-Glo™ or NADP/NADPH-Glo™ Assays. (4945)

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Cell Death Differ. 8, e2526. AIF-independent parthanatos in the pathogenesis of dry age-related macular degeneration. 2017

Jang, K.H., Do, Y.-J., Son, D., Son, E., Choi, J.-S. and Kim, E.

Notes: ARPE-19 cells were seeded at 1 × 104 cells per well in 96-well plates and incubated for 12 hours. Cells were treated with  H2O2 with or without olparib for 4 hours. Cellular NAD+ levels were measured with the NAD/NADH-Glo™ Assay.  Cellular ATP levels were measured with the CellTiter-Glo® Assay. (28292900)

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Biochem. Biophys. Res. Commun. 482, 802–7. Anti-apoptotic quinolinate phosphoribosyltransferase (QPRT) is a target gene of Wilms’ tumor gene 1 (WT1) protein in leukemic cells 2017

Ullmark, T., Montano, G., Järvstråt, L., Jernmark, Nilsson H., Håkansson, E., Drott, K., Nilsson, B., Vidovic, K., Gullberg, U.

Notes: The NAD/NADH-Glo™ Assay was used to assess whether or not overexpression of QPRT in K562 cells would increase cellular NAD levels and that was responsible for QPRT-associated resistance to imatinib. QPRT is in the de novo pathway for NAD synthesis from tryptophan. NAD levels did not rise upon QPRT upregulate but the decreased. (4829)

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114, E2709–8. Hormone and receptor interplay in the regulation of mosquito lipid metabolism. 2017

Wang, X., Hou, Y., Saha, T.T., Pei, G., Raikhel, A.S., and Zou, Z.

Notes: Six mosquitos per sample were frozen in liquid nitrogen and ground in a basic pH lysis buffer then heated to remove NAD+ then neutralized before measuring NADH with the NAD/NADH-Glo™ Assay System. To examine the effect of HNF4 on VLCAD and 3KCT gene expression, the authors employed gel shift assays and luciferase assays of the promoters in pGL4.10[luc2] and control with pGL4.73[hRluc/SV40] transfected into Drosophila S2 cells. Luciferase activities were measured with the Dual-Luciferase® Reporter Assay System on a GloMax® Instrument.  (4834)

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Cancer Prev. Res. 10, 255–66. Loss of BRCA1 in the cells of origin of ovarian cancer induces glycolysis: A window of opportunity for ovarian cancer chemoprevention. 2017

Chiyoda, T., Hart, P.C., Eckert, M.A., McGregor, S.M., Lastra, R.R., Hamamoto, R., Nakamura, Y., Yamada, S.D., Olopade, O.I., Lengyel, E. and Romero, I.L.

Notes: The ratio of NADP+/NADPH was monitored upon BRCA1 knockdown in IOSE397 cells and FT33 fallopian tubes cells using the NADP/NADPH-Glo™ Assay.  The effect of BRCA1 knockdown on the HK2 promoter was evaluated in HEK293T cells using the Dual-Luciferase® Reporter Assay System. (4846)

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Cancer Prev. Res. 10, 255-266. Loss of BRCA1 in the Cells of Origin of Ovarian Cancer Induces Glycolysis: A Window of Opportunity for Ovarian Cancer Chemoprevention. 2017

Chiyoda, T., Hart, P. C., Eckert, M. A., McGregor, S. M., Lastra, R. R., Hamamoto, R. and Romero, I. L.

Notes: In this study, the NADP/NADPH-Glo™ Assay was used to measure NADPH/NADP+ 48 hours after siRNA transfection. (4886)

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Mol. Metab. 6, 535–47. NNT reverse mode of operation mediates glucose control of mitochondrial NADPH and glutathione redox state in mouse pancreatic β-cells. 2017

Santos, L.R.B., Muller, C., de Souza, A.H., Takahashi, H.K., Spégel, P., Sweet, I.R., Chae, H., Mulder, H. and Jonas, J.C.

Notes: Mouse islets were prepared from the pancreas by collagenase digestion and density gradient centrifugation, and then cultured for 2–5 days. The redox state of islets were assessed using batches of 20 islets preincubated for 40 minutes in 0.5mmol/L glucose before treating islets with 2–30mmol/L glucose for 15 minutes. The reaction was stopped and the cells sonicated before measuring NAD+, NADH, NADP+, and NADPH using the NAD/NADH-Glo™ and NADP/NADPH-Glo™ Assays. (4947)

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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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PLoS Genet. 13, e1006590. Re-wiring of energy metabolism promotes viability during hyperreplication stress in E. coli.  2017

Charbon, G., Campion, C., Chan, S.H.J., Bjørn, L., Weimann, A., da Silva, L., Jensen, P.R. and Løbner-Olesen, A.  

Notes: The NAD/NADH-Glo™ Assay was used to determine NAD/NADH ratios in E. coli using a referenced method for extraction. (4832)

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Cancer Res. 77, 4102–15. The alkylating chemotherapeutic temozolomide induces metabolic stress in IDH1-mutant cancers and potentiates NAD+ depletion-mediated cytotoxicity. 2017

Tateishi, K., Higuchi, F., Miller, J.J., Koerner, M.V.A., Lelic, N., Shankar, G.M., Tanaka, S., Fisher, D.E., Batchelor, T.T., Iafrate, A.J., Wakimoto, H., Chi, A.S. and Cahill, D.P.

Notes: Cell viability of IDH1 wild-type or mutant cell lines under treatment conditions was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. From these numbers, the IC50 values were calculated. To evaluate apoptosis, 7,000–8,000 cells were treated with DMSO or compounds singly or in combination (12.5nmol/L NAMPT inhibitors and/or 200μmol/L temozolomide or 50μmol/L Z-VAD-FMK) for 6 or 24 hours and then measured using Caspase-Glo® 3/7 Assay. Changes in NAD+, NADH, NADP+, and NADPH were assessed using the NAD/NADH-Glo™ and NADP/NADPH-Glo™ Assays. (4946)

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Exp. Neurol. 290, 63–73. The neuroprotective compound P7C3-A20 promotes neurogenesis and improves cognitive function after ischemic stroke 2017

Loris, Z.B., Pieper, A.A. and Dietrich, W.D. 

Notes: P7C3 compounds improve the production of NAD through the salvage pathway. NAD levels in rat brain homogenates were monitored with the NAD/NADH-Glo™ Assay using a referenced modification to the protocol. (4830)

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30, 940-952.  Leveraging an NQO1 bioactivatable drug for tumor-selective use of poly (ADP-ribose) polymerase inhibitors.  2016

Huang, X., Motea, E.A., Moore, Z.R., Yao, J., Dong, Y., Chakrabarti, G., Kilgore, J.A., Silvers, M.A., Patidar, P.L., Cholka, A. and Fattah, F.

Notes: NAD/NADH-Glo™ Assay was used to assess metabolic activity in cells. (4897)

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45, 6048-6077. 1001 lights: luciferins, luciferases, their mechanisms of action and applications in chemical analysis, biology and medicine 2016

Kaskova, Z. M., Tsarkova, A. S. and Yampolsky, I. V. 

Notes: This article referenced the NADP/NADPH-Glo™ Assay when describing products for bioluminescent analysis. (4885)

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Cell Death Differ. 23, 1873–85. FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration.  2016

Yu, C., Kim, B.S. and Kim, E. 

Notes: Mouse embryonic fibroblasts were treated with vehicle or DPQ for 1 hour followed by exposure to H2O2.  Intracellular NAD+ was measured with the NAD/NADH-Glo™ Assay and cellular ATP was measured with the CellTiter-Glo® Viability Assay. (4845)

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Front Endocrinol 7, 5. Fenofibrate induces ketone body production in melanoma and glioblastoma cells. 2016

Grabacka, M.M., Wilk, A., Antonczyk, A., Banks, P., Walczyk-Tytko, E., Dean, M., Pierzchalska, M. and Reiss, K. 

Notes: B16 F10 murine melanoma cells were treated with fenofibrate and the ratios of NADP+/NADPH and GSH/GSSG were examined after PPARa knockdown or over-expression. The NADP+/NADPH levels did not change appreciably but the GSH/GSSG levels did fluctuate. The study used the NADP/NADPH-Glo™ and GSH/GSSG-Glo™ Assays. Assays were read on a GloMax® 20/20 Luminometer. (4852)

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Clin. Can. Res. 22, 4452-65. Myc-driven glycolysis is a therapeutic target in glioblastoma.  2016

Tateishi, K., Iafrate, A.J., Ho, Q., Curry, W.T., Batchelor, T.T., Flaherty, K.T., Onozato, M.L., Lelic, N., Sundaram, S., Cahill, D.P., Chi, A.S. and Wakimoto, H.

Notes: CellTiter-Glo® Cell Viability Assay was used to assess the vulnerability of myc-driven cancers to inhibitors of the NAMPT. Sensitivity was correlated to the level of myc expression. Apoptosis was assessed with the Caspase-Glo® 3/7 Assay. The NAD/NADH-Glo® Assay was used to provide a quantitative measure of NAD+ in the cells under study. (4835)

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