Abstract
High-content screening of kinase inhibitors is important in order to identify biogenesis and function mechanisms of subcellular organelles. Here, we present a human kinome siRNA high-content screen on primary human umbilical vein endothelial cells, that were transfected by electroporation. The data descriptor contains a confocal fluorescence, microscopic image dataset. We also describe an open source, automated image analysis workflow that can be reused to perform high-content analysis of other organelles. This dataset is suitable for analysis of morphological parameters that are linked to human umbilical vein endothelial cell (HUVEC) biology.
Highlights
Background & SummaryMicroscopy-based high-content screening has emerged as a powerful method to interrogate biological pathways and to identify novel genes involved in cellular processes[1]
Weibel-Palade bodies (WPB) are the endothelial storage organelles of von-Willebrand factor, a large adhesive glycoprotein that is released upon vascular injury and promotes the recruitment of platelets to the site of injury, thereby initiating primary haemostasis
WPBs can be visualised in cells by von-Willebrand factor (vWF) immuno-staining and factors that regulate this WPB formation can be identified by high-content screening approaches
Summary
Microscopy-based high-content screening has emerged as a powerful method to interrogate biological pathways and to identify novel genes involved in cellular processes[1]. We have previously monitored the length of WPBs in cells by high-throughput morphometry and identified a role for the Golgi ribbon in determining the size of these structures. When assessing the reproducibility among biological replicates, we observed a high level of variability between replicates that might reflect technical limitations of the electroporation Another possibility is that the low dynamic range observed in this assay reflects inherent features of the underlying biology, e.g. that multiple kinases may only have a minor impact on WPB formation. Aspect ratio is a shape descriptor of a WPB, means the ratio of the length of the primary and secondary axes of a WPB’s fitted ellipse. We developed an image analysis pipeline in ImageJ and identified several kinases that regulate the area, number, length and intensity of WPB objects. We present an image analysis workflow for the identification and analysis of key features of cellular organelles, in this case WPBs, that can be applied to other sub-cellular structures in high- or low-throughput settings
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