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PLos ONE 11, e0148433. Regulation of nuclear receptor Nur77 by miR-124  2016

Tenga, A., Beard, J.A., Takwi, A., Wang, Y.-M., and Chen, T. 

Notes: The effect of miR-124 on the Nur77-3’ UTR was analyzed with a dual-reporter plasmid and luciferase activities were measured with the Dual-Glo® Luciferase Assay System. Daoy cells used in the study were transfected with the reporter plasmid and miRNA expression vectors using the FuGENE® 6 Transfection Reagent. FuGENE® 6 was also employed to transfect 293T cells for recombinant lentivirus construction. RNA isolation of mRNA expression level determination by probe-based RT-qPCR was accomplished with a Maxwell® 16 Research Instrument using the Maxwell® 16 LEV simplyRNA Tissue Kit. Viability assays of 2D cultures of Daoy cells were determined with the CellTiter-Glo® Cell Viability Assay. Viability assays of 3D microspheroid cultures of Daoy cells (presumably ULA plate-derived) were measured with the CellTiter-Glo® 3D Cell Viability Assay. (4816)

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Nat Chem. Biol. 12(9), 672–679. Sensitivity and engineered resistance of myeloid leukemia cells to BRD9 inhibition. 2016

Hohmann, A.F., Martin, L.J., Minder, J.L., Roe, J.S., Shi, J., Steurer, S., Bader, G., McConnell, D., Pearson, M., Gerstberger, T., Gottschamel, T., Thompson, D., Suzuki, Y., Koegl, M. and Vakoc, C.R.

Notes: The BRD9 subunit of the SWI-SNF chromatin-remodeling complex is investigated in the context of aberrant cell proliferation in acute myeloid leukemia. Specifically, the NanoBRET system was used to measure the interactions of BRD9 and Histone H3.3. A small molecule inhibitor of the BRB9 bromodomain function, BI-7273, was further assessed in HEK293T cells. Disruption of BRB9 interaction with Histone H3.3 was confirmed at sub-micromolar concentrations of BI-7273. Cell viability in the presence of inhibitor was determined using the CellTiter-Glo System. (5079)

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7, 124. SIRT Is Required for EDP-Mediated Protective Responses toward Hypoxia-Reoxygenation Injury in Cardiac Cells. 2016

Samokhvalov, V., Jamieson, K.L., Fedotov, I., Endo, T., and Seubert, J.M.

Notes: The authors of this study examined the effects of EDPs on LPS-induced toxicity. They considered whether EDPs protect HL-1 cardiac cells from hypoxia-reoxygenation, via SIRT-1 associated preservation of mitochondrial quality. Sirtuins1 enzymatic activity was measured in the whole cell lysates by using SIRT-Glo™ Assay, and caspase 3/7 activity was assessed using the Apo-ONE™ Homogeneous Caspase-3/7 Assay. (5228)

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Biochem. Biophys. Rep. 6, 24-31. Splice variant insertions in the C-terminus impairs YAP’s transactivation domain. 2016

Finch-Edmondson, M.L., Strauss, R.P., Clayton, J.S., Yeoh, G.C. and Callus, B.A.

Notes: A luciferase-based mammalian two-hybrid system was assembled in HeLa and D645 cells using the ViaFect™ Transfection Reagent (1µg DNA/well; 12-well plates). The results of the experiments were read with the aid of the Dual-Luciferase® Reporter Assay System. (4662)

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Antimicrob. Agents Chemother. 60, 4972–82. Targeting the cytochrome bc1 complex of Leishmania parasites for discovery of novel drugs. 2016

Ortiz, D., Forquer, I., Boitz, J., Soysa, R., Elya, C., Fulwiler, A., Nilsen, A., Polley, T., Riscoe, M.K., Ullman, B., and Landfear, S.M. 

Notes: The hRLuc gene from the pGL4.70[hRLuc] Vector was used as the source of Renilla luciferase used to make stably transfected Leishmania mexicana or donovani cell lines. The Renilla luciferase expression was used to monitor parasite loads  of infected cells. Luciferase activity was measured with the Renilla Luciferase Assay System. Amastigotes were treated with endochin-like quinolones and the ratio of NAD/NADH was measured with the NAD/NADH-Glo™ Assay.   (4837)

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JCl Insight 1, e88814. The airway epithelium undergoes metabolic reprogramming in individuals at high risk for lung cancer.  2016

Rahman, S.M., Ji., X., Zimmerman, L.J., Li, M., Harris, B.K., Hoeksema, M.D., Trenary, I.A., Zou, Y., Qian, J., Slebos, R.J., Beane, J., Spira, A., Shyr, Y., Eisenberg, R., Liebler, D.C., Young, J.D. and Massion, P.P. 

Notes: BEAS2B human bronchial epithelial cells were cultured for months in the presence of cigarette smoke condensate. The cells showed metabolic reprogramming including changes in the NADPH/NADP+ ratio. The measurement was made with the NADP/NADPH-Glo™ Assay and read on a GloMax® Instrument.  (4855)

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J. Immunol. 196, 106–14. The immune-metaboic basis of effector memory CD4+ T cell function under hypoxic conditions. 2016

Dimeloe, S., Mehling, M., Frick, C., Loeliger, J., Bantug, G.R., Sauder, U., Fischer, M., Belle, R., Develioglue, L., Tay, S., Langenkamp, A. and Hess, C.

Notes: The NADP/NADPH-Glo™ Assay was used to assess cellular utilization of the pentose phosphate pathway through monitoring NADPH levels. Use of a glucose-6-phosphate dehydrogenase inhibitor, 6-AN, effectively reduced NADPH levels. Data are shown in the supplemental material.  (4854)

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Metabolies 6, E33. The redox status of cancer cells supports mechanisms behind the Warburg effect   2016

Moreira, J.D., Hamrz, M., Abolhassani, M., Bigan, E., Pérès, S., Paulevé, L., Noqueira, M.L., Steyaert, J.M. and Schwartz, L. 

Notes: The NAD/NADH-Glo™, NADP/NADPH-Glo™ and ENLITEN™ ATP Assays were used to monitor differences in ATP levels as well as NAD/NADH and NADP/NADPH ratios in primary  normal and cancerous colon cells in the various stages of the cell cycle. Each assay used 2 ×105 cells per well and were normalized to total protein. (4826)

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Biochem. Biophys. Rep. 6, 1-8. The relationship between RUVBLI (Pontin, Tip49, NMP238) and BCL6 in benign and malignant human lymphoid tissues. 2016

Baron, B.W., Baron, R.M., and Baron, J.M.

Notes: HEK 293T cells were transfected with a reporter plasmid (assembled in the pGL3 Basic Vector) with a consensus BCL6 binding site and a BCL6 expression plasmid using the ViaFect™ Transfection Reagent (about 2µg of DNA per well in 6-well plates at 50-60% confluency). Promoter activation was monitored with a luciferase assay control with a β-galactosidase control. Western blotting of transfected cells used the Anti-Rabbit IgG (Fc), Alkaline Phosphatase Conjugate and the Western Blue® Stabilized Substrate for Alkaline Phosphatase. (4657)

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ACS Synth. Biol. 5, 1376–1382. The signal sequence of the abundant extracellular metalloprotease PPEP-1 can be used to secrete synthetic reporter proteins in Clostridium difficile. 2016

Oliveira Paiva, A.M., Friggen, A.H., Hossein-Javaheri, S. and Smits, W.K.

Notes: The authors fused the PPEP-1 signal sequence to a codon-optimized luciferase gene based on NanoLuc® Luciferase (such as the pNL1.1 Vector) to create a secreted luciferase reporter for C. difficile. Measurements of luciferase activity were performed with 100µl 1:100 sample (either whole cell lysate or culture supernatant) in triplicate in a 96-well plate with 20µl NanoGlo® Luciferase Assay System. (4716)

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Cell Rep. 16, 37–47. The TIP60 complex is a conserved coactivator of HIF1A. 2016

Perez-Perri, J.I., Dengler, V.L., Audetat, K.A., Pandey, A., Bonner, E.A., Urh, M., Mendez, J., Daniels, D.L., Wappner, P., Galbraith M.D. and Espinosa, J.M.

Notes: HaloTag® Pull-Down Assay
HEK293T (12 × 106 cells) were plated and grown to 70–80% confluence (approximately 18 hours). The cells were then transfected (using FuGENE® HD Transfection Reagent [Cat.# E2311]) with either 30µg of HaloTag(HT)-HIF1A or HT-alone control vector (vectors available by custom order from Promega Custom Assay Services). Clarified lysates from both HT-HIF1A and HT-alone control cells were prepared and incubated with HaloLink™ Resin (HaloTag® Mammalian Pull-Down System [Cat.# G6500, G6504]). Proteins were digested with trypsin, and digestion was quenched with formic acid. Digested peptides were analyzed by mass spectrometry.

NanoBRET™ Assay
HCT116 and HEK293 cells (8 ×105) were plated in each well of a 6-well plate and co-transfected with one of three acceptors: HT-Pontin, HT-Reptin or HT-TIP60, in combination with the HIF1A-NanoLuc(NL) donor. The following NanoBRET pairs used are available by custom order from Promega Custom Assay Services: HIF1α-NLuc + HT-TIP60, HIF1α-NLuc + HT-Pontin or HIF1α-NLuc + HT-Reptin. (4718)

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Sci. Rep. 6, 33235. The Use of a Novel NanoLuc -Based Reporter Phage for the Detection of Escherichia coli O157:H7 2016

Zhang, D., Coronel-Aguilera, C.P., Romero, P.L., Perry, L., Minocha, U., Rosenfield, C., Gehring, A.G., Paoli, G.C., Bhunia, A.K. and Applegate, B.

Notes: This study used  E. coli O157:H7 bacteriophage ΦV10 modified to express NanoLuc® luciferase (Nluc) to give a detectable signal from E. coli O157:H7. (4923)

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Nat. Commun. 7, 11392. Thiol reductive stress induces cellulose-anchored biofilm formation in Mycobacterium tuberculosis  2016

Trivedi, A., Mavi, P.S., Bhatt, D. and Kumar, A. 

Notes: Mycobacterium tuberculosis grown as a biofilm or planktonic cells were found to have similar NAD/NADH and NADP/NADPH levels. The ratios were measured with the NAD/NADH-Glo™ and NADP/NADPH-Glo™ Assays. (4828)

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Oncogene , (Epub ahead of print). Topoisomerase IIα mediates TCF-dependent epithelial-mesenchymal transition in colon cancer. 2016

Zhou, Q., Abraham, A.D., Li, L., Babalmorad, A., Bagby, S., Arcaroli, J.J., Hansen, R.J., Valeriote, F.A., Gustafson, D.L., Schaack, J., Messersmith, W.A. and LaBarbera, D.V.

Notes: Topoisomerase IIα was identified as an important factor for metastasis of colorectal cancer involving the wnt signaling pathway. Microspheroids of SW620 cells, generated through ultra-low attachment plates, were used in the study. Activation of the wnt pathway was monitored with a reporter assay measured with the ONE-Glo™ Luciferase Assay System. Duplicate cells were monitored for viability with the CellTiter-Glo® 3D Cell Viability Assay. An ATP-competitive, N-terminal inhibitor of topoisomerase IIα was examined for cytotoxicity to the microspheroids. Cytotoxicity was monitored from 6–72 hours of exposure to the inhibitor using the CellTox™ Green Cytotoxicity Assay. Cytotoxicity increased over time with the inhibitor, and controls were essentially unaffected. (4708)

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Nat. Med. 22, 262–9. Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition. 2016

Hata, A.N., Niederst, M.J., Archibald, H.L., Gomez-Caraballo, M., Siddiqui, F.M., Mulvey, H.E., Maruvka, Y.E., Ji, F., Bhang, H.C., Radhakrishna, V.K., Siravegna, G., Hu, H., Raoof, S., Lockeerman, E., Kalsy, A., Lee, D., Keating, C.I., Ruddy, D.A., Damon, L.J., Crystal, A.S., Costa, C., Piotrowska, Z., Bardelli, A., Iafrate, A.J., Sadreyev, R.I., Stegmeier, F., Getz, G., Sequist, L.V., Faber, A.C. and Engelman, J.A.

Notes: This study investigated the reason why cancers become resistant to anti-cancer treatments over time. Non-small-cell lung cancers were cultured for weeks in the presence of EGF receptor inhibitors and resistant clones obtained based on a specific mutation in the EGF receptor. Clones that became resistant within 6 weeks were found to result from cells already containing the EGFR mutation. Clones taking about 24 weeks to become resistant evolved from the EGFR mutation. RealTime-Glo™ MT Cell Viability Assay was employed in several studies over a 10-week period to monitor proliferation (data presented in Supplementary Figures). Each week prior to media change, RealTime-Glo™ Assay reagents were added, incubated for 1 hour and luminescence measured. After 72-hour incubations, short-term viability was measured with the CellTiter-Glo® Luminescent Cell Viability Assay. (4699)

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Purinergic Signal. [ePub ahead of print]. UDP-glucose promotes neutrophil recruitment in the lung 2016

Sesma, J.I., Weitzer, C.D., Livraghi-Butrico, A., Dang, H., Donaldson, S., Alexis, N.E., Jacobson, K.A., Harden, T.K., Lazarowski, E.R.

Notes: ATP levels in lung secretions from CF patients were quantified by a luciferin-luciferase assay, using the GloMax® Discover System. (4748)

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Integr. Med. Res. 5(2), 131–9. Ulmus davidiana ethanol extract inhibits monocyte adhesion to tumor necrosis factor-alpha-stimulated endothelial cells. 2016

Lee, K.M., Hoo, H.K., Lee, Y.R., Park, M.S., Kang, G., Choi, S., Lee, K.H. and Jeon, B.H.

Notes: An ethanol extract of the Asian Elm tree was examined. To ensure that the extract did not cause nonspecific cell death, HUVEC cells were cultured with various concentrations of the extract and monitored for changes in viability over a 24-hour period. The RealTime-Glo™ MT Cell Viability Assay reagents were added at dosing and viability monitored at desired time points; only the 24-hour time point is shown. (4700)

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Haematologica 101, 1469–78. Uncoupling of the Hippo and Rho pathways allows megakaryocytes to escape the tetraploid checkpoint.  2016

Roy, A., Lordier, L., Pioche-Durieu, C., Souquere, S., Roy, L., Rameau, P., Lapierre, V., Le Cam, E., Plo, I., Debili, N., Raslova, H. and Vainchenker, W. 

Notes: Human megakaryocytes, with control or knockdown YAP, were analyzed for NAD+/NADH with the NAD/NADH-Glo™ Assay. (4843)

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Cell Death Dis. 7, e2073. X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity 2016

Evans, M.K., Sauer, S.J., Nath, S., Robinson, T.J., Morse, M.A. and Devi, G.R.

Notes: The authors demonstrate that the X-linked inhibitor of apoptosis (XIAP) drives anti-EGFR and anti-HER antibody resistance in inflammatory breast cancer both through its effect on caspase and suppression of ROS accumulation. They use the CytoTox ONE Homogeneous Membrane Integrity Assay to assess cytotoxicity in wild type SUM149 and SUM190 cells, and  rSUM149 and rSUM190 cells (cell lines that have acquired resistance to treatment agents) upon treatment with cytotoxic agents. The Caspase-Glo® 3/7 Assay was used to assess apoptosis in wild type cells, rSUM149 and in cells over expressing XIAP (treated and untreated). (4726)

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Mem Inst Oswaldo Cruz. 111, 287–93. Zika virus damages the human placental barrier and presents marked fetal neurotropism 2016

Noronha, Ld., Zanluca, C., Azevedo, M.L., Luz, K.G. and Santos, C.N.

Notes: The authors demonstrate evidence of transplacental transmission of Zika virus through detection of viral proteins and viral RNA in placental tissue samples from expectant mothers infected at different stages of gestational. To confirm the identity of the flavivirus in the IHC assays, the corresponding FFPE tissue block was punched with a hollow needle, and tissue cores 3 mm in width were removed for molecular studies. Total RNA was extracted from these cores using the ReliaPrepTM FFPE total RNA Miniprep System according to the manufacturer’s recommendations. RNA was eluted in 50μL of elution buffer, and 5 μL of the extracted RNA was amplified by real-time RT-PCR using two primer/probe sets specific for ZIKV (Lanciotti et al. 2008). Real-time assays were performed using the GoTaq Probe 1-Step RTqPCR System. (4719)

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J. Med. Chem. 58, 2718–36. 9H-Purine Scaffold Reveals Induced-Fit Pocket Plasticity of the BRD9 Bromodomain. 2015

Picaud, S., Strocchia, M., Terracciano, S., Lauro, G., Mendez, J., Daniels, D.L., Riccio, R., Bifulco, G., Bruno, I. and Filippakopoulos, P.

Notes: The authors used bioluminescence resonance energy transfer (BRET) to test the ability of a bromodomain 9 ligand to disrupt binding to histone. HEK 293 cells were cotransfected with a histone H3.3-HaloTag® fusion vector and either NanoLuc®-BRD9 bromodomain or NanoLuc®-full-length BRD4 fusion vector. After 24 hours, the transfected cells were trypsinized, diluted in phenol red-free DMEM with or without 10nM of HaloTag® NanoBRET™ 618 Ligand and dispensed into a 96-well plate. One of two potential BRD-disrupting compounds, 7d or 11, was adding to a final concentration of 0.005–33μM, cells were incubated for 18 hours and NanoBRET™ Nano-Glo® Substrate (final concentration 10µM) was added. Fluorescence was measured and a corrected BRET ratio calculated. Cytotoxicity was assessed after the NanoBRET™ assay by incubating the cells with the CellTiter-Glo® Reagent for 30 minutes and measuring luminescence. To examine histone H3.3 localization, HEK 293 cells were transfected with the histone H3.3-HaloTag® fusion vector using FuGENE® HD Transfection Reagent. After 24 hours, cells were labeled with 5μM HaloTag® TMR ligand for 15 minutes before washing with complete medium, incubated for 30 minutes and imaged with a confocal microscope. (4568)

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Nat. Commun. 17, 10237. A generic strategy for CRISPR-Cas9-mediated gene tagging 2015

Lackner, D.H., Carré, A., Guzzardo, P.M., Banning, C., Mangena, R., Henley, T., Oberndorfer, S., Gapp, B.V., Nijman, S.M., Brummelkamp, T.R. and Bürckstümmer, T.

Notes: The authors present an overall strategy for using the CRISPR/Cas9 system for reporter tagging of endogenous loci. As examples, NanoLuc® and TurboGFP-tagged reporter cell lines were generated. PCR was performed using GoTaq® Polymerase. To detect the integration event, we combined a primer binding in the cassette (NanoLuc® or TurboGFP) with a gene-specific primer. Cells were analysed using the NanoGlo® Luciferase Assay. (4755)

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Assay Drug Dev. Technol. 13 (8), 444-455. A Homogenous Bioluminescent System for Measuring GTPase, GTPase Activating Protein, and Guanine Nucleotide Exchange Factor Activities. 2015

Mondal, S., Hsiao, K., and Goueli, S.A.

Notes: These authors describe a new bioluminescent assay for monitoring GTPase activity, and demonstrate it's utility for monitoring GTPase, GAP-stimulated GTPase, GAP, and GEF activities. The system can also be used to analyze these proteins expressed in cells as fusion proteins using a pulldown assay format. The assays showed minimal false hits upon testing for compound interference using the library of pharmacologically active compounds. (4584)

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Anal. Biochem. 489, 1–8. A luminescent assay for real-time measurements of receptor endocytosis in living cells. 2015

Robers, M. B., Binkowski, B. F., Cong, M., Zimprich, C., Corona, C., McDougall, M., Otto, G., Eggers, C. T., Hartnett, J., Machleidt, T., Fan, F. and Wood, K. V.

Notes: The authors describe a new method for real-time analysis of ligand-mediated receptor endocytosis in living cells. They used a BRET-based assay to detect the interactions of ligands with various GPCRs fused to NanoLuc® luciferase. (4693)

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Sci. Rep. 5, 17946. Aflibercept traps galectin-1, an angiogenic factor associated with diabetic retinopathy. 2015

Kanda, A., Noda, K., Saito, W. and Ishida, S.

Notes: RealTime-Glo™ MT Cell Viability Assay was used to demonstrate the anti-proliferative properties of aflibercept (a chimeric glycoprotein consisting of the ligand-binding elements of VEGFR1 and VEGFR2 fused to the human IgG1 Fc) on galactin-1- and VEGF-A-stimulated human retinal microvascular endothelial cells (HRMEC). The authors do not indicate the length of the incubation or whether an end-point or real-time monitoring method was used. (4701)

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