Steven Haney

Rapid Assessment and Visualization of Normality in High-Content and Other Cell-Level Data and Its Impact on the Interpretation of Experimental Results

When investigators monitor effects on a population of cells following a perturbation, these events rarely occur in a classical normal (or Gaussian) distribution. A normal distribution is, however, explicitly assumed for events within a single well, in which mean values per well are used as an assay metric and, in general, measures of assay robustness, such as the Z’ score and the V factor. Such analysis is not possible for many technologies; however, high-content screening (HCS) measures events of individual cells, which are averaged over the well. These individual cell-level measurements may be analyzed separately. This study quantifies the extent of nonnormality in experimental samples and their effects on determining the EC50 of a test compound and the assay robustness statistics. The results, based on five sets of publicly available data, indicate that the Z’ or V-factor score can be improved by as much as 0.44 more than standard calculations, and the EC50 of a dose–response curve can be lowered by as much as fivefold when nonparametric methods are used, but not all data sets show a significant improvement. The effect on analysis depends in part on whether the greatest shift from normality occurs in the upper or lower range of the dose–response curve.

A Kinome-Wide siRNA Screen Identifies Multiple Roles for Protein Kinases in Hypoxic Stress Adaptation, Including Roles for IRAK4 and GAK in Protection against Apoptosis in VHL−/− Renal Carcinoma Cells, Despite Activation of the NF-κB Pathway

Hypoxia induces changes to cancer cells that make them more resistant to treatment. We have looked at signaling pathways that facilitate these changes by screening the human kinome for effects on hypoxic responses in SW480 colon cancer cells. Hits identified in the screen were examined for effects on multiple molecular responses to hypoxia, including the endoplasmic reticulum stress and DNA damage responses in colon, melanoma, and renal cancer lines. To validate the hits from the small interfering RNA studies, we developed cell lines expressing stable short hairpin RNAs (shRNAs) in the A498 renal carcinoma cell line. Several lines, including those expressing shRNAs against DYRK1B, GAK, IHPK2, IRAK4, and MATK, showed an inability to form spheroid cultures. In addition, shRNAs targeting IRAK4 and GAK were incapable of 2D growth under anoxia. In the GAK shRNA-expressing line, nuclear factor–κB (NF-κB) was localized to the nucleus, but in the IRAK4 shRNA line, NF-κB levels were increased but the extent of nuclear localization was unchanged. Dominant negative mutants of IRAK4 and GAK also showed strong apoptotic effects in A498 cells under anoxia, supporting a direct link between these kinases and survival of the VHL−/− RCC line, which is typically highly resistant to hypoxic stress as a result of high and constitutive levels of Hif-1α.

An introduction to high content screening : imaging technology, assay development, and data analysis in biology and drug discovery

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High-Content Screening Approaches That Minimize Confounding Factors in RNAi, CRISPR, and Small Molecule Screening

Screening arrayed libraries of reagents, particularly small molecules began as a vehicle for drug discovery, but the in last few years it has become a cornerstone of biological investigation, joining RNAi and CRISPR as methods for elucidating functional relationships that could not be anticipated, and illustrating the mechanisms behind basic and disease biology, and therapeutic resistance. However, these approaches share some common challenges, especially with respect to specificity or selectivity of the reagents as they are scaled to large protein families or the genome. High-content screening (HCS) has emerged as an important complement to screening, mostly the result of a wide array of specific molecular events, such as protein kinase and transcription factor activation, morphological changes associated with stem cell differentiation or the epithelial-mesenchymal transition of cancer cells. Beyond the range of cellular events that can be screened by HCS, image-based screening introduces new processes for differentiating between specific and nonspecific effects on cells. This chapter introduces these complexities and discusses strategies available in image-based screening that can mitigate the challenges they can bring to screening.