Notes: The role TGF-β in neovascular age-related macular degeneration (nAMD) is presently debated. Here, the levels of active TGF-β1, β2 and β3 proteins were measured in aqueous humor of patients with nAMD. The Nano-Glo® Dual-Luciferase® Reporter was then used to assess TGF-β pathway activation in Lenti-X 293T cells treated with aqueous humor from patients. Patients never treated with ranibizumab showed lower pathway activation compared to control or treated patients. Data here suggests a protective effect of TGF-β2 on nAMD. (5094)

Notes: Infection progression remains poorly characterized in Chagas disease, a life-long infection caused by T. cruzi. To understand the development of the disease, a bioluminescent imaging system for live mice infected with NanoLuc-labeled transgenic T. cruzi was developed. Initial expression of NanoLuc luciferase was determined in TcCOL cells and cell lysates using the Nano-Glo Live Cell assay and the Nano-Glo Luciferase Assay System, respectively. Luminescence could be detected as early as 14 days post-infection and was focused in the abdomen. Further, luminescence remained detectable for up to 222 days post infection, while parasite number in blood decreased after 21 days to the limit of detection.  (5110)

Notes: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are both neurodegenerative diseases caused by hexanucleotide repeat expansions. The data presented here implicates a model were accumulation of dipeptide repeat proteins (DPR) using non-AUG translation leads to stress induction and disease progression. The Nano-Glo Dual Luciferase assay is utilized with both monocistronic and bicistronic reporters to assess canonical AUG translation and RAN translation. RAN translation of these dipeptide repeat proteins does not require the AUG start codon, 5’-cap or EIF4e. (5127)

Notes: Tandem repeats (TMs) are thought to be unstable genetic elements, however the presence of regulator binding motifs with TMs may allow them to function as cis-regulatory elements. ZEB1, a transcriptional repressor which promotes mesenchymal maintenance, was shown to occupy TR regions near loci relevant to epithelial identity. The minimum number of repeat regions for ZEB1 binding and transcriptional repression was determined using the Nano-Glo Dual Luciferase Assay System. ZEB1 motifs within TRs were cloned upstream of the NanoLuc reporter. Relative luminescence was measured, and repression was correlated with motif number. (5124)

Notes: Common and dimer-specific DNA binding sites were determined for interferon regulatory factors (IRF3, IRF5, and IRF7). Only IRF3 and IRF7 bound to virus-response elements (VRE) of interferon regulated genes. Mutational analysis of IRFs showed a single residue affected specificity for VRE binding, which was absent in IRF5. The Nano-Glo Dual Luciferase Reporter was used to determine transcriptional activation of IRF variants. (5125)

Notes: The authors identified single nucleotide mutations in gene regulatory regions that may result in transcriptional changes in the context of lung adenocarcinoma. Further, 31 of these regulatory region mutations were found in clinical lung adenocarcinoma isolates and were associated with patient outcomes. The Nano-Glo® Dual-Luciferase® Reporter Assay was used to compare expression with wild-type or mutant regulatory regions. Specifically, the relative activity of the mutant NFATC1 regulatory region was shown to be 3-fold higher compared to wild-type. (5092)

Notes: MicroRNAs function in post-transcriptional regulation of gene expression by binding target mRNAs and influencing translation. Here, let-7 microRNA translational activation is observed for mRNAs lacking poly-A tails in a cell-free system. The Nano-Glo Dual Luciferase assay was used to validate activation of capped but non-adenylated mRNAs in HEK293T cells. Interestingly, both PABP and PAPD7 were involved in the observed translational stimulation. Together, this presents a model were polyadenylation influences microRNA-based activation or repression of target mRNA. (5126)

Notes: Ribavirin, displayed efficacy against Rabies virus in vitro, however could not be translated clinically. To address this, novel ribavirin analogs were developed and tested for antiviral activity and cytotoxicity. (5109)

Notes: An insertional mutagenesis screen of the Dengue virus genome was used to identify regions tolerating small 15 amino acid insertions. The NS1 protein, which is essential for viral genome replication, was shown to be highly tolerant to insertions. NS1 was tagged with both a minimal fusion tag (Small BiT) and NanoLuc Luciferase. Tagged protein variants were assessed for viral infectivity, and used to investigate the protein localization and levels of NS1. (5075)

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)

Notes: The role of Polycomb-repressive complex 1 (PRC1) in spermatogenesis is investigated. RNF2, a component of PRC1, together with SALL4 is shown to bind the transcription start sites (TSSs) of genes involved in spermatogenesis. Further, a direct interaction between SALL4 and RNF2 is shown using coimmunoprecipitation. To validate a direct role of the SALL4-RFN2 complex in gene activation, regulatory elements were placed upstream of the NanoLuc luciferase and luminescence was monitored in a range of cell types. Together, a mechanism of gene activation is present where RNF2 activates transcription of sall4, and collectively they act on target genes. (5132)

Notes: The interplay of heat shock factor 1 (HSF1) and inhibition of mRNA splicing in response to heat shock is investigated. SF3B1 is identified as a regulator splicing during heat shock. Using the Nano-Glo Dual Luciferase assay, small molecule inhibition of SF3B1 showed inhibition of the heat shock response. A HSF1-Gal4 DNA binding domain fusion was co-transfected with a vector containing a Firefly reporter with Gal4 DNA binding element. HSF1 activation directly influenced firefly luciferase expression though the Gal-4 element. (5133)

Notes: The interplay of Vestigial and Scalloped proteins was determined in the context of Drosophila wing and muscle development. The function of Vestigial in specific cell types is shown to be dependent on both phosphorylation and the presence of Scalloped. Nano-Glo® Dual-Luciferase® Reporter Assay was used to assess activation of an enhancer element in the presence of Vestigial and Scalloped proteins. Mutations in Vestigial at phosphorylation sites show a decrease in enhancer element activation. (5093)

Notes: The authors characterize a novel orange-red fluorescent protein (CyOFP1), which serves as an efficient acceptor for resonance energy transfer from NanoLuc^® luciferase. An optimized fusion protein of CyOFP1 and NanoLuc^® (Antares) was demonstrated to function as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. During fluorometry, 50,000 cells in 10 μL Live Cell Imaging Solution were loaded into glass capillary tubes and gently pelleted by centrifugation for 15 s at 500g. 10 μL of Live Cell Imaging Solution with 20 μM D-luciferin, coelenterazine, ViviRen, or furimazine was then added. For BRET6 and Antares-expressing mice, tail vein injections were performed with 333 nmol (123 μg) of furimazine in 120 μL of 8% glycerol, 12% ethanol, 10% hydroxypropyl-β-cyclodextrin, and 35% polyethylene glycol 400 in water. (4760)

Notes: NanoLuc^® and Firefly luciferase reporters (pCMV128-NanoLuc and pCI-FireflyLuc) were co-transfected into HeLa cells to provide complementary measures of specific mRNA splicing and export activity. In the presence of splicing activity, firefly luciferase pre-mRNA is spliced in the nucleus, exported and translated resulting in firefly luciferase activity. In contrast, the open reading frame of the Nanoluc luciferase in pCMV128 NanoLuc^® was placed between two weak splice sites. If the pre-mRNA is spliced, the NanoLuc coding sequence is removed and no luciferase can be expressed. The NanoLuc^® reporter can only be translated when the unspliced pre-mRNA is transported to the cytoplasm. pCMV128 NanoLuc^® was generated by inserting the NanoLuc^® sequence into the NotI and BamHI sites of pCMV128, and then used for export assays. Cells were harvested 72 h post-transfection using 300 μl Passive Lysis Buffer. Lysates were analyzed using the NanoGlo® Dual-Luciferase^® Reporter Assay. (4744)

Notes: NanoLuc® Luciferase was used to monitor Plasmodium berghei throughout it's life cycle, including detecting single parasites in mosquitos, livers and asexual blood stages. (4922)

Notes: The CellTiter-Glo® 3D Cell Viability Assay was used to measure viability of MDA-MB-468 cells grown on Matrigel with inducible knockdown of PNUTS. The effect of PNUTS knockdown was also assessed in a reporter model of E-Cadherin expression using a firefly luciferase reporter vector and Renilla luciferase control vector. Luciferase activities were measured with the Dual-Luciferase® Reporter Assay System. (4823)

Notes: A NanoLuc^® luciferase reporter was used to further understand the mechanism of Nrf2 inhibition of proinflammatory cytokine gene induction. Approximately 1 kb upstream region of the IL6 gene TSS containing the WT ARE (GCTGAGTCA) or mutated ARE (AGATCTGAC) was conjugated to the translation start site of the NanoLuc^® gene in the pNL2.2 vector. 293T cells (2.0 × 10³ cells per well) were plated in 96-well plates 24 h before transfection. The NanoLuc^® reporter vectors were co-transfected with pQC-FLAG-hNRF2T80R (constitutively active NRF2) plasmid33 and pcDNA3-p65 plasmid. After 48 hours of the transfection, the luminescence was quantified and normalized using NanoGlo^® Dual-Luciferase^® Reporter Assay. (4752)

Notes: These authors describe use of firefly and NanoLuc® luciferases in a coincidence reporter system to screen compounds for their ability to activate transcription of the Parkin gene (PARK2), a potential target for treatment of Parkinson's disease. In a coincidence reporter system, a single transcript containing two luciferase genes is generated. Inclusion of a 2A “ribosomal skipping” sequence between the two luciferase genes enables translation of two reporter proteins. This approach is useful in drug screening because the use of two independent reporters to monitor a single transcriptional event provides a way to distinguish true “hits” from experimental artifacts caused by interaction between library compounds and reporter proteins. True “hits” (compounds that affect transcription of the gene being monitored) cause a signal from both reporters; false positives caused by interaction of compounds with the reporter protein cause a signal from only one reporter. This paper describes the first use of firefly and NanoLuc® luciferases, and the Nano-Glo® Dual-Luciferase® Reporter Assay, in a coincidence reporter system to screen a compound library in an HTS assay. (4565)

Notes: These authors studied the interactions between G-protein coupled receptors (GPCRs)  and 13 different G-proteins using a NanoBRET assay. NanoBRET assays were performed using protein partners labeled with NanoLuc luciferase or Venus yellow fluorescent protein. The improvement in signal-to-noise ratio achieved using the NanoBRET method enabled resolution of signals from previously intractable G-proteins and GPCRs. The authors demonstrated that GPCRs engage multiple G-proteins with distinct patterns of activity or “fingerprints”. This differential engagement of multiple target G-proteins was revealed by quantitative analysis of G-protein activation kinetics.  (4588)

Notes: The authors investigated the interaction between androgen receptors and miRNAs in order to better understand the role of miRNAs in prostate cancer biology. A NanoLuc^® and firefly luciferase miRNA target expression vector (pmirNanoGlo) was used to evaluate the predicted role of miR-22 and miR-29a in the regulation of target genes LAMC1 and MCL1. This bicistronic vector contains NanoLuc^® luciferase (NlucP) as the primary reporter gene and Firefly luciferase (Luc2) as the control reporter for normalization. For each target gene, the cDNA fragment predicted to encompass miR-22 and miR-29a target sites, was inserted into multiple cloning regions located in the 3′-UTR of the NanoLuc^® reporter vector. PC3 cells were transfected with the reporter vectors in combination with either miR-22 mimic, miR-29a mimic and the Negative Control 4 or antimiR-22, antimiR-29a, and the Negative Control. Transfected PC3 cells underwent NanoLuc^® and Firefly luciferase activity measurements using the NanoGlo^® Dual-Luciferase® Assay. (4753)

Notes: This paper introduces NanoBRET technology, which provides an improved alternative to conventional BRET protein interaction assays. NanoBRET assays combine the extremely bright NanoLuc luciferase with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The greater light intensity and improved spectral resolution of the NanoBRET assay results in increased detection sensitivity and dynamic range over current BRET technologies. Performance of the assay is demonstrated using several model systems, and the ability to image BRET in individual cells is illustrated. The  authors also demonstrate the application of NanoBRET in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells. (4575)

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)