Combining High-Content Imaging and Phenotypic Classification Analysis of Senescence-Associated Beta-Galactosidase Staining to Identify Regulators of Oncogene-Induced Senescence

Assay Drug Dev Technol. 2016 Sep;14(7):416-28. doi: 10.1089/adt.2016.739.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Transformed
  • Cellular Senescence / physiology*
  • High-Throughput Screening Assays / methods*
  • Humans
  • Oncogenes / physiology*
  • Phenotype*
  • Staining and Labeling / methods*
  • beta-Galactosidase / analysis*


  • beta-Galactosidase