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- Title
Combining High-Content Imaging and Phenotypic Classification Analysis of Senescence-Associated Beta-Galactosidase Staining to Identify Regulators of Oncogene-Induced Senescence.
- Authors
Chan, Keefe T.; Hannan, Ross D.; Pearson, Richard B.; Paavolainen, Lassi; Horvath, Peter; Hannan, Katherine M.; George, Amee J.; Simpson, Kaylene J.
- Abstract
Hyperactivation of the PI3K/AKT/mTORC1 signaling pathway is a hallmark of the majority of sporadic human cancers. Paradoxically, chronic activation of this pathway in nontransformed cells promotes senescence, which acts as a significant barrier to malignant progression. Understanding how this oncogene-induced senescence is maintained in nontransformed cells and conversely how it is subverted in cancer cells will provide insight into cancer development and potentially identify novel therapeutic targets. High-throughput screening provides a powerful platform for target discovery. Here, we describe an approach to use RNAi transfection of a pre-established AKT-induced senescent cell population and subsequent high-content imaging to screen for senescence regulators. We have incorporated multiparametric readouts, including cell number, proliferation, and senescence-associated beta-galactosidase (SA-βGal) staining. Using machine learning and automated image analysis, we also describe methods to classify distinct phenotypes of cells with SA-βGal staining. These methods can be readily adaptable to high-throughput functional screens interrogating the mechanisms that maintain and prevent senescence in various contexts.
- Subjects
CELLULAR aging; BETA-galactosidase; CELL communication; CELL proliferation; TUMOR suppressor proteins; DNA damage
- Publication
Assay & Drug Development Technologies, 2016, Vol 14, Issue 7, p416
- ISSN
1540-658X
- Publication type
Article
- DOI
10.1089/adt.2016.739