Gene expression profiling after inhibition of Erg in HUVECs. Microarray analysis of differential gene expression in HUVECs was performed at 24 and 48 hours after Erg inhibition by GeneBloc. (A) Composite plot of intensity profiles of the comparison between control and Erg-deficient HUVECs at 24 hours. Genes with > 1.3-fold up-regulation (red) or down-regulation (green) are shown. (B) Analysis of putative gene function for Erg-regulated genes using the DAVID functional annotation and clustering tool reveals enrichment for genes associated with cell migration, organization of the actin and microtubule cytoskeleton, and GTPase regulation. (C) The ROCK software was used to generate a functional interaction network based on the list of Erg-regulated migration hits (see supplemental Table 2). Gene nodes colored in blue are down-regulated, red are up-regulated, and white are nonhit connectors. Black lines represent direct physical interactions; green lines represent complex or indirect interactions. (D) Comparison between microarray datasets was carried out by Gene Set Enrichment Analysis (GSEA).¹⁶  The dataset of differentially regulated genes after 24 hours of Erg inhibition in HUVECs was compared with the dataset of genes up-regulated by Erg overexpression in a non-EC line (HEK293).¹⁴  GSEA was performed using standard settings with 1000 data permutations. The graphical outputs show enrichment (green curve) of the Erg overexpression gene set along ranked lists of all genes (middle). Below is the full Erg dataset ranked according to intensity. This analysis identified a significant inverse correlation between the genes down-regulated after Erg inhibition in HUVECs and those up-regulated after Erg overexpression in HEK293 cells. Normalized enrichment score (NES) = −1.3540292, P < .001.