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P2033Targeting & manipulating the STAT, M1-NFAT, & NFêB signaling pathways in quantitative high-throughput screening (qHTS) using a laser-scanning fluorescence microplate cytometer
Presenter Natasha Thorne, NIH Chemical Genomics Center, USA
Additional Authors: Natasha Thorne, Jennifer Wichterman, Ronald L. Johnson, Noel Southall, Christopher P. Austin, Doug Auld and James Inglese
The transcription factors STAT, NFAT, and NF?B regulate the expression of a multitude of genes with key roles in cellular growth and differentiation, tissue development, and immune system activation and response. Unregulated or constitutively active forms of these transcription factors leads to cellular transformation and oncogenesis. Inhibiting components of these signaling pathways thus has great therapeutic potential. Using cellular-based assays with a ß-lactamase reporter gene as transcriptional read-out, we perform qHTS to assess the activity of thousands of small molecules against these transcription factors. In this way, we are able to identify small molecules that down-regulate ß-lactamase expression, indicating inhibition of transcription factor activation or function. The commonly used high-throughput technology for ß-lactamase reporter gene assays relies on population-plate readers (such as PerkinElmer’s EnVision), in which aggregate fluorescence from thousands of cells is used to quantitate response to a small molecule. While providing robust output, this type of reader may lack sensitivity, particularly when only a fraction of the cells respond to stimulus. An alternative technology is the Acumen laser-scanning fluorescence microplate cytometer, which provides information on individual cells - ideal for reporter assays in which cells may have varying responses to transcriptional stimulation or inhibition. Preliminary experiments indicate that the Acumen may not only provide a more accurate and sensitive assessment of the activity of small molecules in cell signaling pathways, but also serve as a platform for addressing exactly how these compounds exert their cellular effects.