Notes: The authors of this paper investigated mutations affecting the hinge loop of protein kinases that appear to confer resistance to both Type I and Type II inhibitors. They introduced individual amino acid substitutions into the hinge region of six distantly related protein kinases and determined the inhibitor sensitivity of these kinases. The ADP-Glo™ Kinase Assay was used to asses the activity of the Haspin and c-Src kinases and the engineered mutants in this study. (4144)

Notes: The authors of this study evaluated the ADP-Glo™ Assay technology for use in high-throughput screening applications for inhibitors of all four known mammalian PI 4-kinases. They found that K_m values, IC₅₀ values of known inhibitors, and dose-response curves were comparable to values reported in the literature or those obtained using the standard isotopic assay. Z´-factor values for the assay in a low-volume, 384-well format were 0.72 and 0.74, indicating that the assay would be suitable for screening activities in 384- or 1536-well formats. (4129)

Notes: This paper describes the use of the ADP-Glo™ Assay technology to screen for small-molecule inhibitors of PI 4-kinases (phatidylinositol 4-kinases). The authors characterized E. coli-expressed proteins using the ADP-Glo™ Kinase Assay and saw strong signal-to-background ratios, no signal from their negative controls or the D1899A protein, and no signal in the absence of micelles. Next they compared data from the ADP-Glo™ Assay with results obtained from the standard isotopic method or results available in the literature. They were able to obtain K_m values for PI4KA and PI4KB that corresponded to published values. Finally, the authors evaluated the potential of the ADP-Glo™ Assay as a high-throughput screening tool. To assess the assay, they used Z´ factor as their statistical measure, which is a description of the dynamic range and the variability of the assay. In this test, the Z´ factor values were 0.72 and 0.74. From these results, the authors conclude that the assay would work well for screening in a 384-well system and could likely be optimized for screening in a 1536-well format. (4135)

Notes: To confirm the role of a mutation in the miR-96 microRNA (miRNA) associated with an autosomal dominant hearing lost, HeLa cells (250,000 cells per well in six-well plates) were transfected with 4µg of plasmid carrying wild type or mutant miR-96 miRNA using FuGENE® HD Transfection Reagent. After 24 hours, the cells were washed and total RNA extracted. After quantitation, the RNA used in RT-PCR analysis. The entire 3´UTRs of eight putative target genes were amplified by PCR from genomic DNA and cloned into the psiCHECK™-2 Vector. HeLa cells were transiently transfected with 2µg of the 3´ UTR psiCHECK™-2 constructs and 0.2µg of a wild-type, single or double mutant miR-96 plasmid using FuGENE® HD Transfection Reagent. Forty-eight hours after transfection, the Dual-Luciferase® Reporter Assay System was used to quantify the firefly and Renilla luciferase in cell lysates. (4251)

Notes: This study characterized interactions between the proton-pumping membrane complex V-ATPase, the Arf nucleotide binding site opener ARNO, and aldolase. The authors used a combination of protein-protein interaction techniques to identify downstream effectors of ARNO and V-ATPase signaling, and identified aldolase as a specific interaction partner of ARNO that could be involved in intracellular trafficking and cytoskeletal modulation. As part of the study, the FluoroTect™ Green_Lys in vitro Translation Labeling System was used to fluorescently label recombinant proteins during in vitro translation reactions. The labeled proteins were used in pull-down experiments. (4250)

Notes: These authors investigated the ability of Phyllanthus plant extracts to affect the metastatic activity of human lung (A549) and breast (MCF-7) cancer cell lines. They initially used CellTiter 96® AQ_ueous Non-Radioactive Cell Proliferation Assay and the GloMax®-Multi Detection System absorbance module to determine cytotoxicity of various Phyllanthus extracts. After determining the effective dose, the authors investigated the ability of these plant compounds to inhibit/reduce metastatic activity. They then evaluated the mechanism of cell death in treated cells using the Caspase-Glo® 3/7 Assay and the Glomax® Multi Detection System luminescence module to measure caspase activity, and the CytoTox-ONE™ Homogeneous Membrane Integrity Assay to measure LDH activity. (4196)

Notes: The authors of this study sought to correlate promoter methylation with gene expression. Gene expression data was generated by RNA-seq of Jurkat cells. Amplified cDNA was prepared from total RNA, the cDNA was treated with S1 nuclease to remove single stranded nucleic acids and used as template for the sequencing libraries. (4536)

Notes: These authors used the Maxwell^® 16 Blood DNA Purification Kit to isolate genomic DNA from leukocytes. (4210)

Notes: Here the authors describe development of an HTS cell viability assay protocol for use with cultured cyropreserved human primary hepatocytes. Cryopreserved hepatocytes for culturing were prepared as suspensions and dispensed at 2,000 or 4,000 cells/5µl/well in collagen I-coated 1,536-well plates. Cells were allowed to attach and then 23nl of each test compound was added in a dilution series from 2.8nM to 92µM, and cells incubated for 24 or 40 hours. Five microliters of CellTiter-Glo^® Reagent was added and cells were incubated 30 minutes before reading the luminescent output. IC₅₀ values for 12 compounds were determined; a summary of the protocol is provided in Table 1 of the paper. Cultured cryopreserved hepatocytes were assayed for function using the P450-Glo^® CYP3A4 assay with the Luciferin-IPA substrate. (4182)

Notes: The authors constructed red light-emitting reporter systems based on bioluminescence resonance energy transfer (BRET) for ratiometric imaging of protein-protein interactions (PPIs) in cell culture and deep tissues of small animals. These BRET systems consist of Renilla reniformis luciferase (RLuc) variants, RLuc8 and RLuc8.6, used as BRET donors, combined with two red fluorescent proteins, TagRFP and TurboFP635, as BRET acceptors. They used the EnduRen™ Live Cell Substrate in their BRET systems to produce a red-shift emission maxima optimal for deep-tissue imaging. To demonstrate this capability, the authors imaged HT-1080 cells accumulating in the lungs of nude mice. The cells expressed BRET fusion proteins in the context of rapamycin-induced FK506 binding protein 12 (FKBP12)-FKBP12 rapamycin binding domain (FRB) association. Mice were injected with luciferase substrate at 1.5 hours after cell injection to produce a bioluminescence image. Their data suggest that the BRET systems could be used for drug screening and target validation applications. (4130)

Notes: The authors describe the modification of the Promega MAO-Glo™ Assay to create a robust high-throughput screen for semicarbazide-sensitive amine oxidases (SSAOs). These proteins are implicated in inflammatory diseases and are believed to play a role in immune cell trafficking and cell activation. Their modified MAO-Glo™ Assay was tested with enzyme from recombinant and cell sources, and was able to identify specific inhibitors. (4155)

Notes: Briefly, BHK-21 cells were plated the day before transfection at a density of 5 × 10⁵ cells into a 100-mm cell culture dish. A total of 19 μg of each plasmid was mixed with Fugene HD reagent and incubated for 10 min at room temperature. Each mixture was added to confluent monolayers of BHK-21 cells and incubated at 37°C in a 5% CO₂ incubator. At 24 hours posttransfection, the cells were lysed. (4427)

Notes: This paper describes a rapid dual-luciferase-based assay for L1 retrotransposition that is amenable to high-throughput screening. A firefly luciferase vector in which the luciferase gene was disrupted by an antisense intron was constructed by introducing a 900-bp fragment of the human γ-globin intron into pGL4.13. This Fluc gene, interrupted by an antisense intron, gives only minimal luciferase expression unless the luciferase gene is restored by a retrotransposition event. The authors also tested a similar retrotransposition reporter using the pGL4.73 Renilla luciferase vector, but found that the firefly construct gave much higher signals. They therefore used the firefly luciferase retrotransposition reporter, a Renilla luciferase normalization control and the Dual-Luciferase^® Assay to characterize profiles of retrotransposition by various human and mouse L1 elements, and to measure the kinetics of L1 retrotransposition in cultured cells. The GloMax^® Multi Luminometer was used to quantify luciferase activity. (4205)

Notes: In this paper, FuGENE® 6 reagent was used to transfect PC3 human prostate cancer cells at a 4:1 FuGENE®:DNA ratio. (4370)

Notes: The authors developed a loop-mediated isothermal amplification (LAMP) assay to distinguish between virulent and nonvirulent strains of Vibrio vulnificus by targeting the virulence-correlated gene (vcg). The authors performed PCR using vcg-specific primers and GoTaq^® Hot Start Polymerase in parallel to confirm the LAMP results. (4163)

Notes: These authors used extensive computational analysis of isolates from the 2009 H1N1 outbreak to identify conserved H1N1 sequence signatures that could potentially be used in diagnostic assays to track the spread of specific strains during viral outbreaks. They identified target sequences in the hemagglutinin and neuraminidase genes that were highly conserved among 2009 H1N1 isolates. They then designed primers to amplify those sequences and used them in Taqman® and SYBR® Green-based qPCR assays to create a discriminatory 2009 H1N1 detection assay. They used the AccessQuick™ System in conventional RT-PCR to first establish whether their chosen primers were specific for the 2009 H1N1 isolates. In that assay they amplified representative H1N1 strains spanning from 1930 to the 2009 H1N1, and showed that only the 2009 isolates generated product. (4284)

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)

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)

Notes: This study analysed the roles of Dcp2 and Nudt16 in nonsense-mediated mRNA decay miRNA-mediated silencing. The authors used various luciferase reporter constructs to evaluate the significance of Dcp2 and Nudt16 in miRNA- and siRNA-mediated gene silencing in wildtype and knockout MEF cells. Various Renilla luciferase constructs containing or lacking miRNA target sites were cotransfected along with a control plasmid encoding firefly luciferase (for normalization purposes). Renilla and firefly luciferase luminescence were measured using the Dual-Luciferase^® Reporter Assay and the GloMax^®-Multi Luminometer. (4204)

Notes: This paper explored potential compounds as agonists of dopamine D2 receptor (D2R) with a bias toward β-arrestin signaling. Based on the aripiprazole scaffold, compounds were synthesized and tested in a D2-mediated Gi-coupled isoproterenol-stimulated cAMP production assay using HEK293T cells expressing D2R transfected with pGloSensor™-22F cAMP Plasmid. Assessing β-arrestin recruitment to agonist-stimulated receptors was determined using HTLA cells stably expressing β-arrestin-TEV protease and a tetracycline transactivator-driven luciferase exposed to agonist or D2 test ligand with or without reference agonist. After 18 hours, medium was removed from the cells, 1X Bright-Glo™ Reagent added and luminescence measured. (4518)

Notes: These authors showed that expression of the serine protease inhibitor elafin is regulated by epigenetically controlled expression of the transcription factor Foxa2. Treatment of melanoma cells with a DNA methyltransferase inhibitor induced elafin expression, resulting in reduced proliferation and increased apoptosis. Luciferase reporter assays were used to show that Foxa2 binding was required for activation of elafin expression, and that Foxa2 binding was activated by treatment with the methyltransferase inhibitor. These assays used a pGL3-Basic Vector construct in which expression of firefly luciferase was driven by the upstream region bearing the Foxa2 binding site. The pRL-TK Vector, expressing Renilla luciferase, was used as a normalization control. The AccessQuick™ System was used for RT-PCR analysis to show that Foxa2 mRNA was barely detectable in melanoma cells.  (4345)

Notes: The authors of this paper investigated the molecular mechanisms through which fumaric acid esters affect the progression and pathology of multiple sclerosis. The authors looked at the activation of the Nrf2 transcriptional pathway which plays a role in defense against oxidative stress. A stable reporter cell line expressing eight copies of the gluthione-S-transferase 2 antioxidative response elements cloned upstream of the luciferase complementary DNA in a pGL4.26 vector. Reporter cells were stimulated with dimethylfumarate for 24 hours and luciferase activity measured using the Luciferase Assay System. A strong dose-dependent induction of antioxidative response elements was observed. Next, the authors looked at the effects of fumaric acid esters on the inhibitor of Nrf2, Keap 1. C-terminal, V5-tagged Keap1 protein was transfected into 293 cells. Forty-eight hours post transfection, cells were treated with dimethyl fumarate. Samples were lysed, Keap1-V5 was immunoprecipitated and gel purified. The protein was digested with trypsin in the presence of ProteaseMAX™ Surfactant, Trypsin Enhancer. Peptide pools were analyzed using liquid chromatography-tandem mass spectrometry. The authors were able to show that the Keap1 protein was modified in response to treatment. (4215)

Notes: The authors used the CellTiter®-Glo Assay to assess the effect of HER2-Affitoxin on cell viability of gastric carcinoma NCI-N87 cells, which overexpress HER2. The HER2-Affitoxin is a fusion protein of a HER2-specific Affibody and modified Pseudomonas aeruginosa exotoxin A (PE 38). This fusion protein was designed to offer an alternative method for treating tumors that do not respond to trastuzumab or have acquired resistance to current therapies. The CellTiter-Glo® Assay was used to calculate HER2-Affitoxin IC₅₀ values of cells in culture. They describe the mechanism of action as follows: HER2-Affitoxin binds to HER2 protein on the cell surface, is internalized, where it blocks protein synthesis in the cytosol via ADP ribosylation of eEF-2. (4131)

Notes: Yuangheng He and colleagues asked how the weak alkaline compound chloroquine (CQ) enhances the anti-inflammatory effects of synthetic glucocorticoids like dexamethasone, which are used to treat a host of inflammatory and autoimmune diseases. In the process they explored the intersection of lysosomal degradation pathways and glucocorticoid receptor signaling. They used Dual-Glo® Luciferase Assay System to look at glucocorticoid receptor-mediated (GR) activation and repression of reporters in AD293 cells under a variety of conditions (presence or absence of CQ; stripped serum, loss of lysosome synthesis, inhibition of V-ATPase, etc). They also used HaloTag® protein fusions to observe the fate of GR populations in the presence or absence of CQ and in the presence or absence of compounds that impair proteasome function. Live-cell imaging of GR-HaloTag® protein fusions revealed a dynamic association of the GR with lysosomes. The authors showed that glucocorticoid signaling is regulated by lysosomes. (4203)

Notes: In this study, caspase-3/7 activity in HUVECs was measured using the Caspase-Glo^® 3/7 Assay. Luminescence was measured with the GloMax^®-96 Microplate Luminometer. (4207)