Figure 1: Overview of the successive siRNA migration screens.
From: Systematic high-content genome-wide RNAi screens of endothelial cell migration and morphology
(a) Experimental scheme for the genome-wide siRNA screen to identify genes required for cell migration. HDLEC cells were reverse transfected with siRNA SMARTpools 48 h prior to assessment for migratory capacity in the monolayer scratch assay. Image based analysis was used to calculate a migration score and a cell density score. The primary screen assessed siRNA SMARTpools targeting 18,120 protein-coding genes (see Data Record 1). (b) Automated image analysis was used firstly for cell nuclei count; samples that were identified as ‘Low Cell Density’ were excluded from migration analysis. Image analysis was then used to measure cell migration (Data Record 4). Results with migration |robust z score|>2 were considered as candidate hits. (c) The deconvolution screen confirmed the phenotypes of 154 genes as regulators of LEC migration (see Data Record 2). (d) A tertiary screen was performed to compare and contrast the role of confirmed migration regulators in two dermal endothelial cell types: HDLECs and HMBECs (Data Record 3). Morphological analysis of gene knockdown phenotypes was also performed in order to identify genes with similar functional roles (Data Records 5 and 6).
