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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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)