Analysis of the effect of shear stress on basal and TNF-α–induced gene expression in endothelial cells. (A) Hierarchical clustering of a selection of genes modulated more than 2-fold by shear, TNF-α, or shear and TNF-α combined; all 3 treatments relative to static control conditions. Three main cluster groups can be discriminated, which were analyzed for the presence of overrepresented TFBSs in the promoters of the genes in each group compared with the whole genome in conserved 500 base pair upstream regions in the human-mouse alignment of May 2004. A selection of transcription factors with overrepresented binding sites is shown for each cluster group, and highlighted in orange are transcription factors that are well described to be involved in inflammatory signaling. (B-D) Real-time PCR validation of gene expression as obtained by microarray. PCR analysis was done for a selection of genes from each of the 3 cluster groups as identified by microarray data analysis. Real-time PCR was performed in duplicate on HUVEC cDNA from 3 independent isolates, comparing static conditions with either 7 days of pulsatile shear stress (19 ± 12 dynes/cm²), a 6-hour treatment of TNF-α (25 ng/mL), or both. Expression levels relative to P0 housekeeping gene were obtained for a selection of genes from each cluster group and represented relative to static controls. Error bars indicate SEM. Significant difference versus static control conditions is indicated (*P < .05; **P < .01); ns indicates not significant.