Notes: The role of miR-19b-1 and its target, VEZF1, in angiogenesis and breast cancer is investigated. Overexpression of miR-19b-1 showed marked decrease in angiogenesis and inhibited vessel imitation. VEZF1 expression in response to miR-19b-1 is measured using the β-galactosidase enzyme assay. Mutations in the 3’UTR of VEZF1 show loss of regulation by miR-19b-1. Together, this data shows miR-19b-1 functions as a tumor suppressor and may be utilized as a biomarker for breast cancer.  (5021)

Notes: Glucocorticoid receptor (GR) plays critical roles in cell growth, inflammation, and gluconeogenesis. Here, the regulation of GR by 14-3-3β and ϒ is characterized. Transcription of GR in the presence of 14-3-3 proteins was monitored by the β-galactosidase Enzyme Assay and the Luciferase Assay System. 14-3-3β and ϒ showed transcriptional activation of both GR, glucose-6-phosphate, and phosphoenolpyruvate carboxylase (PEPCK).  (5120)

Notes: These authors investigated the effects of various anesthetics on bioluminescence imaging with firefly luciferase. They observed decreases in luminescence with volatile anaethetics, and found increased luciferase expression with injectable anaethetics in intact cells, but not in cell lysates in vitro. They concluded that the decreases in luciferase activity observed with volatile anaesthesia were due to hemodynamic effects, and not due to a direct inhibitory effect on luciferase enzyme itself. The apparent enhancement of luciferase activity with certain injectable anaesthetics appeared to be due to cytotoxic effects that resulted in increased permeablity to luciferase, as the same enhancement was not observed in cell lysates. D-Luciferin was used for in vivo imaging experiments. The pGL4.10 vector (encoding firefly luciferase), Luciferase Assay Reagent, and the GloMax® 96 Microplate Luminometer were used for in vitro assays using cell lysates. (4190)

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: 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: 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)

Notes: The pGL3-Control Vector was co-transfected into COS-7 cells with mammalian expression vectors expressing either heat shock protein Hsp70 or Hsp105α.  The cells were ATP-depleted in glucose-free media with 2μM rotenone and 5mM 2-deoxyglucose for up to 3 hours. Afterwards the cells were lysed with the Cell Culture Lysis Reagent and assayed for luciferase activity uaing the Luciferase Assay System. ATP depletion was verified during 2μM rotenone and 5mM 2-deoxyglucose treatment using the CellTiter-Glo^® Assay.  (2841)

Notes: The interleukin (IL)-1ß stimulated release of granulocyte macrophage colony stimulating factor (GM-CSF) from lung epithelial cells was explored in this study. To test the promoter activity of GM-CSF, the promoter and enhancer regions were amplified by PCR from human peripheral blood mononuclear cells and cloned into the pGEM^®-T vector. After verification by sequencing, the promoter and enhancer were cloned individually and together into the pGL3-Basic Vector. Additionally, an Xho I/Sal I fragment containing the HSV tk promoter, a gene conferring neomycin resistance, and a poly-A tail were cloned into the Sal I site of pGL3 to allow the production of stable transfectants. To perform stable transfections, 20µl of the Tfx™-50 transfection reagent was incubated with 8µg of plasmid in serum-free medium for 15 minutes at room temperature.  Preconfluent human A549 type II alveolar carcinoma cells were incubated with the transfection mix for 2 hours after washing with serum-free medium. The cells were then cultured in fresh medium for 16 hours before the addition of  0.5mg/mL G-418. After 21 days, foci of cells developed and were harvested for use in luciferase assays. The cells were plated into 24-well plates, grown to confluency and incubated in serum-free medium. Stimulation by 1ng/ml IL-1ß or 1µM PMA for 12 hours was followed by lysate production and measurement of luciferase activity using the Luciferase Assay System and a Turner luminometer. Luciferase readings were standardized against total protein measurements. The PolyATract^® System IV was also used prior to a Northern Blot to obtain purified mRNA. (2742)