Notes: The significant loss of estrogen in women after menopause has been linked to increased incidence of Alzheimer’s disease. Here, the protective effect of β-ecdysterone (β-Ecd) on SH-SY5Y cell apoptosis in relation to Alzheimer’s disease is investigated. NF-κB and estrogen receptor activation was monitored in the presence of β-Ecd in Aβ-stress conditions using the Dual-Glo® Luciferase Assay System. Caspase activation was measured as a proxy for cell stress and apoptosis. Together, SH-SY5Y cell treatment with β-Ecd showed a protective effect, making β-Ecd a promising therapeutic candidate. (5169)

Notes: Expression and regulation of Δ6 fatty acyl desaturase (Fads2) is compared in carnivorous marine fish and salmonids. A novel regulatory region with several binding sites for transcription factors was identified in Epinephelus coioides. Interestingly, the regulatory binding site for stimulatory protein 1 (Sp1) was absent. Truncations of the 5’ UTR were analyzed for changes in expression using the Dual-Glo® Luciferase Assay System. (5170)

Notes: miR-195 overexpression is shown to repress non-small cell lung cancer (NSCLC) tumor growth through a high-throughput screen using the CellTiter-Glo® Assay. To determine miR-195 targets, regulatory regions of prospective targets were cloned into a luciferase fusion construct and monitored using the Dual-Glo® Luciferase Assay System. miRNA was shown to regulate cyclin D3 and survivin, leading the regulation of cell cycle arrest and apoptosis. (5171)

Notes: These authors studied the role of the antioxidant transcription factor nuclear factor E2-related factor-2 (Nrf2) in adaptation to inflammatory/environmental stress in malignant colonic epithelial cells. They used the Dual-Glo® Luciferase Assay and pGL3-Basic Vector in reporter assays, and the CellTiter® 96, Caspase-Glo® 3/7 and Glucose Uptake-Glo™ Assays to investigate Nrf2 effects on cell viability and metabolism. They found that Nrf2 has an impact on the metabolism in premalignant colonic epithelial cells exposed to inflammatory M1 macrophages, an effect accompanied by growth and survival alterations.  (5014)

Notes: These authors suggest that claudin-2, which is highly expressed in human lung adenocarcinoma cells and is involved in the up-regulation of cell proliferation, may be a novel therapeutic target in lung adenocarcinoma. Claudin-2 knockdown increased the accumulation of anticancer agents in cancer cells and spheroids. As part of the study, the CellTiter-Glo® 3D Assay was used to assess viability of Claudin-2KD/A549 spheroid cultures. (5021)

Notes: Serine/Glycine biosynthesis was examined for a panel of 79 non-small cell lung carcinomas, and each could be classified into a high-synthetic and a low-synthetic group based on expression of four enzymes involved in serine/glycine synthesis from glucose. ATF4 was identified as a key mediator of NRF2 activity for serine/glycine biosynthesis through transcriptional activation and not regulation of ATF4 translation (the latter being examined in a dual-luciferase reporter assay using Dual-Glo® Luciferase Assay System). A byproduct of the conversion of serine to glycine is production of NADPH. Seven high-synthetic cell lines exhibited a marked decrease in NADPH levels with regard to NADP+ level when the rate-limiting serine/glycine synthetic enzyme was knocked down with shRNAs with little change in NADPH/NADP+ ratios in six low-synthetic cell lines. The levels of NADPH and NADP+ were monitored with the NADP/NADPH-Glo™ Assay. Expression of the various shRNAs used in the study did not induce cytotoxicity as judged by staining with the CellTox™ Green Cytotoxicity Assay and visual examination by fluorescent microscopy. The identity of all cell lines used in this study were verified by STR analysis with a PowerPlex® System. (4715)

Notes: The putative promoter and deletion mutants of the proposed promoter of the RBHG gene were cloned into the pGL3-Basic Vector for reporter assay investigation. Reporter plasmids and the pRL-TK Control Vector were cotransfected into HepG2 cells with the ViaFect™ Transfection Reagent (transfection details not provided). Forty-eight hours post-transfection, reporter activity was measured with the Dual-Glo® Luciferase Assay System using a GloMax® Instrument. (4685)

Notes: For construction of the Snai1 30 UTR reporter, the CMV promoter was subcloned into the promoterless pGL3-Basic vector upstream of the luciferase gene. A 755-bp Snai1 30 UTR fragment containing miR-133a-binding sites was amplified by PCR and subcloned into the modified pGL3-Basic vector. The activities of firefly luciferase and renilla luciferase in the control vector were determined by the Dual-Glo^® Luciferase Assay System. RNA was extracted from MEFs, GMT-, GMT/miR-133-, or GMT/miR-133/ Snai1-induced aMHC-GFP+ cells, neonatal mouse heart tissues, HCFs, GMTMM-, GMTMM/miR-133-, GMTMM/miR-133/Snai1-transduced HCFs using ReliaPrep™ RNA Cell Miniprep System for gene microarray analysis. (4737)

Notes: To understand the mechanism of Panicle blast 1 (Pb1) gene-mediated resistance to rice blast, a rice fungal disease, researchers investigated Pb1 interacted with a transcription factor involved in resistance, WRKY45 that is regulated by the ubiquitin system. To study how these proteins interacted, inner rice leaf sheaths were bombarded with gold particles coated with 0.5 µg of effector plasmid, 0.3 µg of NanoLuc® luciferase reporter and 0.1 µg of reference Renilla luciferase. After incubating overnight at 28°C, samples were ground in liquid nitrogen and reporter activities assayed using the Dual-Glo® Luciferase Reporter Assay System and Nano-Glo® Luciferase Assay System. The Renilla luciferase gene was also split into an N-terminal construct and C-terminal construct, expressed in rice protoplasts and assayed for reconstituted Renilla luciferase activity. Expression was normalized to firefly luciferase. (4510)

Notes: To better understand what roles FAM123A may play in signaling, cell behavior and human disease, HT1080 sarcoma cells were plated on MatTek dishes coated with 5mg/ml fibronectin before transfection with Venus (a yellow fluorescent protein)-tagged FAM123A or Venus-WTX, another member of the FAM123 gene family, using FuGENE® HD Transfection Reagent. The cells were imaged the next day for low-resolution analysis. For a higher magnification, dynamic analysis, HT1080 cells were plated onto Delta T dishes coated with 5mg/ml fibronectin and transfected with EGFP-FAM123A and FuGENE® HD Transfection Reagent. The next day, cell images were captured every 10 seconds. Examining the effect of silencing FAM123A, a reporter assay used the pGL4.34[luc2P/SRFRE/Hygro] Vector cotransfected with a CMV-Renilla coreporter and other effector plasmids or siRNA, and luciferase levels assessed using the Dual-Glo® Luciferase Assay System. (4246)

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: The authors used a biochemical assay using Xenopus egg extracts to monitor degradation levels of two Wnt pathway components, Axin and ß-catenin, and identify modulators of the Wnt pathway. ß-catenin and Axin were expressed in vitro as firefly and Renilla luciferase fusion proteins, respectively, using the TNT^® SP6 High-Yield Protein Expression System and shown to behave in Xenopus extracts in a similar way to wildtype proteins, which were expressed as radiolabled proteins in rabbit reticulocyte lysate. Degradation of labeled proteins was monitored by SDS polyacrylamide gel electrophoresis and autoradiography, and degradation of luciferase fusion proteins was examined using the Dual-Glo^® Luciferase Assay. Using the Xenopus extract-based assay, the authors screened chemical libraries to identify two modulators of the Wnt pathway, then confirmed this inhibition in HEK 293 cells by demonstrating a decrease in ß-catenin expression with increasing concentrations of the inhibitors. This decrease in ß-catenin-Fluc levels was measured using the Steady-Glo™ Luciferase Assay, and luciferase activity was normalized to cell viability, as determined using the CellTiter-Glo^® Assay. (4181)

Notes: The authors used Dual-Glo^® Luciferase Assay to measure a pISG54 promoter-driven firefly luciferase gene (0.5µg) and pRL-TK plasmid constitutively expressing Renilla luciferase (0.1µg), and either a plasmid (0.5µg) expressing the corresponding variants of Ebola virus (EBOV) structural protein VP24 construct (phCMV-EBOV-VP24) or empty phCMV in HEK 293T and GPC-16 transfected cells. A total of 1.1µg of DNA was transfected. Cells were stimulated with interferon (IFN) 24 hours post-transfection, harvested 16 hours later, and assayed for dual-luciferase activity. Data indicated that mutations in the V24 protein were associated with EBOV virulence but that this virulence was not linked to the IFN-antagonist function of V24 protein. (4178)

Notes: The authors identified a single nucleotide polymorphism (SNP) in the 3´ untranslated region (3´ UTR) of MDM4, which promotes tumorigenesis by decreasing p53 tumor suppressor function, in ovarian cancer cells. This A to C transversion creates a putative target site for the hsa-miR-191 microRNA in the MDM4-C allele, but not the wildtype MDM4-A allele. To determine if this SNP affects MDM4 translation efficiency or mRNA stability, the authors cloned a 224-bp fragment of the MDM4 3´UTRs into the psiCHECK™-2 Vector and transfected the ovarian cancer cell line A2780 with the 224A or 224C variants of the MDM4 3´ UTR. The authors used the luciferase-based psiCHECK™-2 Vector and Dual-Glo^® Luciferase Assay System to show that the C variant dramatically reduces translation efficiency and/or mRNA stability. They also assessed MDM4 expression levels in A/A, A/C and C/C ovarian cancer cells and tissues using RT-qPCR. qPCR primers for MDM4 were designed using the Plexor^® Primer Design Software, and assays were performed using the Plexor^® qPCR System. (4157)

Notes: The authors investigated the role of pioglitazone on transcriptional regulation of the apoA-I gene and looked at the biological properties of pioglitazone-induced apoA-I-containing high-density lipoprotein particles (HDL). To investigate the biological properties of the HDL particles, the authors treated THP-1 cells with conditioned medium from HepG2 cultures treated or untreated with pioglitazone and looked at adhesion to human aortic endothelial cells (HAEC). During the experiment, HAEC viability and proliferation were monitored using the CellTiter-Glo^® Luminescent Cell Viability Assay. Additionally, to determine whether pioglitazone stimulates apoA-I transcription, a luciferase reporter construct was made containing the apoA-I gene promoter. Transfected cells were treated with pioglitazone, and luciferase expression was monitored using the Dual-Luciferase^® Reporter Assay System. (4173)

Notes: To examine post-transcriptional regulation of B cell lymphoma (bcl)-2 mRNA in human cell lines, the bcl-2 open reading frame was cloned into the pCI-neo Mammalian Expression Vector with a FLAG tag. This construct was transfected into U2OS human osteosarcoma cells, lysed, immunoprecipitated on Anti-FLAG-coated beads and analyzed by SDS-PAGE. A 260bp fragment containing the human c-myc 3´UTR adenine-uridine(AU)-rich element (ARE) was cloned into the pGL4.71 [hRlucP] Vector above to produce the pGL4.71PmARE plasmid. For transient expression, SK-N-BE and HEK293 cells in 96-well plates were cotransfected with 200ng of the pGL4.71 constructs (carrying c-myc or bcl-2 ARE) and 200ng of the pGL3-Control Vector. Luciferase expression was assessed using the Dual-Glo^® Luciferase Reporter Assay System. HEK293 cells were also stably cotransfected with the same pGL4.71 constructs and a vector carrying G418 resistance. Renilla luciferase expression was assessed and clones chosen with similar expression levels. (4071)

Notes: In this paper, the role of prostaglandin E2 (PGE2) stimulation of integrin-linked kinase (ILK) in human lung carcinoma was explored. Mutations of Sp1 and NF-κB cis-acting elements in an ILK promoter-pGL3-Basic Vector construct were created using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The mutations were confirmed via sequencing. Human non–small cell lung carcinoma (NSCLC) cells were plated at a density of 5 × 10⁵ cells per well in six-well plates and transfected with 2µg of ILK promoter reporter vectors with or without 0.2µg of the phRL-TK Renilla Luciferase Reporter Vector. After 24 hours, the transfected cells were exposed to PGE2 and the cells lysed for assessment using the Dual-Luciferase^® Reporter Assay System. NSCLC cells were transfected with inactive (ILK-S343A) and superactive ILK (ILK-S343D) cDNA, incubated for 24 hours, treated with or without exogenous PGE2 or with an Sp1 inhibitor for 2 hours. The numbers of viable cells were measured using the CellTiter-Glo^® Luminescent Cell Viability Assay. (4026)

Notes: HepG2 cells in a T-25 culture flask (3 million cells at 70ndash;80% confluency) were transfected with CYP3A4 reporter plasmid (a pGL3 reporter construct) and a CMV-Renilla control plasmid (a total of 2.5µg of plasmid mix was used) using FuGENE® 6 Reagent for transient transfection assays. Reporter activities were measured using the Dual-Glo® Luciferase Assay System. (4268)

Notes: These authors used the Flexi^® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag^® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi^® System and Gateway^® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag^® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)

Notes: In this study, the Renilla luciferase gene from the phRL-CMV Vector was incorporated into polioviral RNA in place of a structural protein-coding region. Renilla luciferase activity was then monitored as a convenient indicator of viral replication. Specifically, these authors tested whether the guanine nucleotide exchange factors GBF1 and BIG2 could rescue brefeldin A (BFA)-induced inhibition of polioviral replication in HeLa cells. Cells were transfected with plasmids encoding either GBF1 or BIG2, together with the firefly-luciferase-encoding pGL4.13 Vector, which was used as a control to monitor transfection efficiency. Eighteen hours post-transfection, the cells were transfected with polioviral replicon RNA containing the Renilla luciferase gene. One hour later, normal growth medium containing EnduRen^® Live Cell Substrate and either BFA or solvent alone, was added and Renilla luciferase activity was monitored at hourly intervals for 7 hours. Cells were then lysed and firefly luciferase activity was measured to assess transfection efficiency using the Dual-Glo^® Luciferase Assay System System. In the presence of BFA, GBF1 was able to partially rescue viral replication. In the presence of BIG2 and BFA, no viral replication occurred. (3562)

Notes: The authors wanted to screen inhibitory compounds for the HIV-1 accessory protein Nef using both computer modeling and experimental methods. Using a structure-based program for the SH3 binding surface of Nef, drug compounds were screened in silico and then further analyzed using a cell-based assay. The Nef gene and SH3 domain were cloned into the pACT and pBIND Vectors of the CheckMate™ Mammalian Two-Hybrid System, transfected into COS-7 cells, and 18 hours later, the cells exposed to potential inhibitors. After 24 hours, luciferase activity was assessed using either the Dual-Glo™ or the Steady-Glo^® Luciferase Assay Systems. (3751)

Notes: To explore the role that accessory proteins may play in successfully expressing odorant receptors (ORs) on the cell surface of heterologous cells, the authors explored cotransfecting receptor-transporting proteins RTP1S, Ric8b and G_αolf with an N-terminally tagged OR to generate a functional and ligand-specific expression system. Three tags (Rho, FLAG or HA) were inserted into the NheI and EcoRI sites of the pCI Mammalian Expression Vector. Amplified OR orfs were cloned into the MluI and NotI sites of the tagged pCI Mammalian Expression Vector. The accessory proteins were subcloned into HA-pCI (RTP1S) or pCI (RTP1S, Ric8b, Hsc70t and G_αolf). For cotransfection, the N-terminal tagged OR vectors (0.8µg) and the accessory proteins (individually or in combination; 0.8µg) were transfected into HEK293T or Hana3A cells. The transfected cells were then subjected to immunocytochemistry, live-cell surface staining, permeabilized staining or FACS analysis. The Dual-Glo™ Luciferase Assay System was used to assess OR activation via CRE elements on a firefly luciferase vector. pRL-SV40 Vector was the internal control for cell viability and transfection efficiency. HEK293T or Hana3A cells were plated on 96-well plates, transfected with 1µg of CRE-Luc vector, 1µg of pRL-SV40 Vector, 5µg of OR and 1µg total for all accessory proteins (0.25µg each protein with pCI Mammalian Expression Vector to keep amount of plasmid constant). Twenty-four hours posttransfection, the medium was changed to CD293 chemically defined medium, incubated for 30 minutes at 37°C then replaced with 25µl of odorant solution in CD293 for a second incubation of 4 hours at 4°C. Then the reporter protein expression levels were measured by luminescence. (3687)

Notes: In this family-based study of 1,231 autism cases, a genetic association of a common C allele in the promoter of the MET gene and autism was identified. Initial sequencing of the MET genes from 86 individuals with autism was used to identify several candidate variants in the MET promoter and 3´ untranslated region. Family-based analyses were then performed to determine whether an association could be demonstrated between any of these variants and autism. A G/C variant 20bp 5´ of the MET transcription start site was found to be overrepresented among individuals with autism, particularly among families where more than on child was affected. Once the candidate variant was identified, the effect of the G/C change on transcription of the MET gene was investigated in a reporter assay. Two 762 bp fragments of the MET promoter region, differing only in the G/C nucleotide, were cloned into pGL4.10 firefly luciferase reporter vectors. These vectors were then transfected into mouse neural cell lines, and luciferase production was monitored using the Dual-Glo™ Assay. The construct containing the C allele produced less than half of the luciferase activity of construct containing the G allele. (3579)

Notes: To screen a compound library for an inhibitor of FoxM1 transcriptional activity, U2OS cells with doxycycline-inducible FoxM1-green fluorescent protein (GFP) fusion protein were stably transfected with a firefly luciferase construct driven by a 6× FoxM1 responsive promoter and the pRL-CMV Vector. These cells were grown overnight in 96-well plates, treated with doxycycline and test compounds. Firefly and Renilla luciferase expression was assessed using the Dual-Glo™ Luciferase Assay System. (3730)