Abstract
Systems-level approaches have emerged that rely on analytical, microscopy-based technology for the discovery of novel drug targets and the mechanisms driving AR signaling, transcriptional activity, and ligand independence. Single cell behavior can be quantified by high-throughput microscopy methods through analysis of endogenous protein levels and localization or creation of biosensor cell lines that can simultaneously detect both acute and latent responses to known and unknown androgenic stimuli. The cell imaging and analytical protocols can be automated to discover agonist/antagonist response windows for nuclear translocation, reporter gene activity, nuclear export, and subnuclear transcription events, facilitating access to a multiplex model system that is inherently unavailable through classic biochemical approaches. In this chapter, we highlight the key steps needed for developing, conducting, and analyzing high-throughput screens to identify effectors of AR signaling.
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Acknowledgments
This work was funded by NIH 5R01DK055622, the Hankamer Foundation, DOD Prostate Cancer Research Program (DAMD W81XWH-10-1-0390) and pilot grant, and equipment support from the John S. Dunn Gulf Coast Consortium for Chemical Genomics (MA Mancini). Additional funding was provided by NIH 1F32DK85979 (SM Hartig), 5T32HD007165 (BW O’Malley), and 5K12DK083014 (DJ Lamb). Imaging resources were supported by SCCPR U54 HD-007495 (BW O’Malley), P30 DK-56338 (MK Estes), P30 CA-125123 (CK Osborne), and the Dan L. Duncan Cancer Center of Baylor College of Medicine. The authors thank the members of the Mancini Lab for thoughtful discussion and J.H. Price (Vala Sciences) and Tim Moran (Accelrys) for longstanding support in automated cytometry.
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Hartig, S.M., Newberg, J.Y., Bolt, M.J., Szafran, A.T., Marcelli, M., Mancini, M.A. (2011). Automated Microscopy and Image Analysis for Androgen Receptor Function. In: Saatcioglu, F. (eds) Androgen Action. Methods in Molecular Biology, vol 776. Humana Press. https://doi.org/10.1007/978-1-61779-243-4_18
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DOI: https://doi.org/10.1007/978-1-61779-243-4_18
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