Egr-3 is required for VEGF-mediated induction of proangiogenic and proinflammatory genes in primary human endothelial cells. (A) Quantitative real-time PCR analysis of Egr-3 mRNA expression in HUVECs transfected with control siRNA (si-Control) or 2 independent siRNAs against Egr-3 (si-Egr-3 oligo1 and -2) and then treated in the presence of 50 ng/mL VEGF for 1 hour. Egr-3 expression is normalized to Cyclophilin A mRNA levels. The results show the mean ± SD of expression relatives relative to si-Control obtained from at least 5 independent experiments. *P < .001 compared with si-Control. (B) Western blot analysis of Egr-3 protein in HUVECs transfected with control siRNA or 2 independent siRNAs against Egr-3 and treated in the absence of presence of 50 ng/mL VEGF for 1 hour. Nucleoporin was used as a loading control. The data are representative of 4 independent experiments. (C) DNA microarrays of HUVECs transfected with si-Control, si-Egr-3 oligo1, or si-Egr-3 oligo2 and treated with 50 ng/mL VEGF for 1 hour. Shown are heat maps of genes whose VEGF response was most profoundly inhibited by si-Egr-3. Transcriptome data were derived from triplicate arrays of VEGF-treated si-Control–transfected cells and duplicate arrays of each of the VEGF-treated si-Egr-3–transfected cells. (D) DNA microarrays of HUVECs transduced with Ad-control or Ad-expressing Egr-1 (Ad-Egr-1) or Egr-3 (Ad-Egr-3; multiplicity of infection = 5). Cells were harvested for RNA at 24 hours after infection. Shown is heat map of siEgr-3–mediated down-regulated genes in Ad-Egr vs Ad-control–transduced cells.