Notes: The relationship of the MAP3K1-JNK and Wnt signaling pathways in embryonic eyelid closure is investigated using a mouse model. Using a MAP3K1-β-galactosidase fusion construct and the Beta-Glo^® Assay System, endogenous levels of MAP3K1 were determined. Additionally, the Dual-Luciferase^® Reporter Assay was used to monitor MAP3K1 protein expression in response to overexpression or downregulation of Wnt. Interestingly, Wnt activation and overexpression showed inhibition of MAP3K1 expression, and Wnt knockouts showed loss of eyelid closure. (5119)

Notes: Mucosal microbiota has been shown to influence HIV-1 infection. Here, P. gingivalis outer membrane vesicles (OMVs) show stimulation of receptor-independent HIV-1 entry into epithelial cells. A secretory NanoLuc luciferase is fused to the nef gene of the HIV genome, cell supernatant is collected, and luciferase activity is measured as a proxy for viral replication. The presence of P. gingivalis vesicles increased viral infectivity approximately 10-fold. (5107)

Notes: Mutations in RAS are commonly associated with pancreatic, colorectal and non-small-cell lung cancers. Treatments targeting RAS in cancers have proven to be difficult due to compensatory resistance mechanisms and upstream pathway insensitivities. Here, SHP2, a protein linking receptor tyrosine kinase signaling to the MAS-RAF-MEK pathway, is shown to lead to cell senescence under growth factor-limiting conditions. The Beta-Glo^® Assay System is used to monitor cell senescence using senescence-associated β-galactosidase, and the CellTiter-Glo^® assay is used to determine cell viability. Together, inhibition of SHP2 showed downstream effects on the RAS signaling pathway and could be sufficient for tumor senescence. (5118)

Notes: The authors used the Beta-Glo® Assay System to determine the transfection efficiency of electroporation. Control of transfection efficiency was done by using a rous sarcoma virus-β-galactosidase expression plasmid at 0.5μg/electroporation. The Plasminogen activator inhibitor-1 (PAI-1) was expressed with a luciferase reporter in a variety of cell lines to show that the effect of insulin to increase PAI-1 is increased by expression of Forkhead-related transcription factors (Fox). To identify which Fox mediated the effects of insulin on PAI expression, small interfering RNAs specific for each Fox were tested along with chromatin cross-linking and ChIP experiments. The authors were able to demonstrate that insulin acts through FoxO3a to increase PAI-1 gene expression, a major regulator of fibrinolysis. (4128)

Notes: The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase. (3926)

Notes: The authors of this paper describe the development of a cell-free assay system derived from Agrobacterium tumefaciens NTL4(pCF218) (pCF372), which expresses β-galactosidase under the control of a tra1 promoter. The tra1 promoter is responsive to quorum-sensing signals. The assay was developed to address time-consuming cell conditioning and incubation times of the standard whole-cell assays used to detect quorum-sensing signals in natural systems. Replacing the X-Gal substrate with the luminescent Beta-Glo^® substrate increased the assay sensitivity by tenfold. (3936)

Notes: TSP50 is a testis-specific gene found to be overexpressed in human breast cancer tissue. Of interest is a putative p53 binding site in the TSP50 promoter. To examine what effect p53 may have on TSP50 expression, the TSP50 promoter was cloned into a pGL3 Luciferase Reporter Vector and cotransfected with a wildtype or R249S mutant p53 and a control vector, pCMV/β-galactosidase, into HeLa, HEK293 and paired MCF7 cells. After 24 hours, the cells were assessed for luciferase expression using the Luciferase Assay System and normalized to β-galactosidase expression, which was measured using the Beta-Glo^® Assay System. (3598)

Notes: PUF proteins act to reduce expression upon binding to the 3´UTR of target mRNA sequences. In this study, the binding of various PUF proteins (including C. elegans FBF-1) to known PUF binding sites was evaluated using a yeast three-hybrid system. For this assay, various PUF proteins fused to the Gal4 activation domain were expressed in pACT and pACT2 plasmids, and DNA oligos expressing PUF binding RNA sequences were cloned into the plasmid pIIIA/MS2-2. Assays were then performed in the yeast strain YBZ-1. β-galactosidase expression, indicating interaction between each PUF protein and the various binding sites, was measured using both solid- (colony lift) and liquid-phase assays. For the liquid-phase assays, β-galactosidase expression was measured using the Beta-Glo^® Assay System. Cells were grown in selective media to an O.D.₆₀₀ of 0.1–0.3, then mixed with an equal volume of Beta-Glo^® Assay Reagent. After one hour, luminescence was measured. Luminescence values were normalized to cell number. The results of these binding assays were used to formulate a consensus PUF binding sequence, which was then used to screen a C. elegans 3´ UTR database for potential FBF-1 target sequences. (3458)

Notes: Methylation of the WTH3 promoter region has been associated with multidrug resistance in breast cancer cells in vitro. In this study, the effect of WTH3 in primary cultured multidrug resistant cells was evaluated, and the influence of a frequently methylated CpG23 region on WTH3 promoter activity was investigated using a luciferase reporter assay system. Promoter regions containing wildtype or mutated CpG regions were cloned into the pGL3 Vector and transfected into paired MCF7 cell lines, along with a control pGL3 Vector without insert, and a plasmid containing the β-galactosidase gene as a transfection efficiency control. Luciferase activity was measured using the Steady-Glo® Luciferase Assay System, and β-galactosidase activity was determined using the Beta-Glo® Assay System. Luciferase activities of the wildtype and mutant promoter regions were compared after normalization to β-galactosidase activity and protein concentration. (3457)

Notes: CEDIA assays (cloned enzyme donor immunoassays) utilize antibodies to specific analytes that can also bind one of the two beta-galactosidase fragments. The beta-galactosidase fragments can associate with each other provided that the fragment bound to the analyte is not immunoabsorbed, thus blocking the fragment interaction.  The Beta-Glo^® Assay System was compared to colorimetric and chemiluminescent detection systems for use in a CEDIA assay with valporic acid as analyte. The Beta-Glo^® Assay System gave the best performance in this assay system.  (3224)

Notes: These authors describe use of the Beta-Glo^® Assay System to analyze beta-galactosidase reporter enzyme concentrations from yeast three-hybrid experiments. The authors optimized the Beta-Glo^® assay with various Saccharomyces cerevisiae strains. The optimal incubation time using the Beta-Glo^® reagent on yeast was one hour, whether the cells were in stationary or exponential growth phase. Data is presented as arbitrary light units from the luminometer. (3295)

Notes: In this paper, the effect of a 5’ untranslated region from the Bcl-2 gene transcripts on firefly and Renilla reporter constructs was evaluated. A number of studies were performed using various single- and dual-reporter constructs containing the Bcl-2 5’ UTR. These constructs were transfected into 293T cells and assayed for luciferase activity using the Dual-Luciferase^® Reporter Assay System. Transfection studies with firefly luciferase mRNA constructs were also performed. In these experiments, firefly luciferase levels were measured using the Luciferase Assay System.  Transfections were normalized using the pSV-β-Galactosidase Control Vector and the Beta-Glo^® Assay System.  (3125)

Notes: The Beta-Glo® Assay System was used to normalize luciferase readings in dual-reporter transfection studies.  In these experiments, human prostate carcinoma PC-3 cells were transfected with a NF-κB factor-driven luciferase reporter construct and a undefined beta-galactosidase reporter construct.  Cells were treated with 20 and 40µM apigenin for 16 h or with 10ng/ml TNF-α for 30 minutes.  Luciferase reporter enzyme expression was assessed with the Luciferase Assay System.  (3054)