Figure 5
Figure 5. ERG binds to the −56 ETS motif. (A) VWF expression is shown as a function of ERG expression levels. Results are based on the Brown datasets (left) and our compendium of 5532 arrays (right). Red, endothelial cells; black, nonendothelial cells. (B) Quantitative real-time PCR analysis of β-actin, CD31, ERG, friend leukemia virus integration-1, and VWF in HUVECs treated with 20 ng/mL TNF-α for 0 (untreated control), 4, 12, or 24 hours. For each gene, expression is shown relative to control values. Data are expressed as mean + SD of 3 independent experiments. (C) ChIP assay was performed using HUVECs treated in the absence of presence of TNF-α for 12 hours. DNA was sheared, and the resulting DNA-protein complexes were immunoprecipitated in the absence or presence of antibodies to ERG or control IgG. Real-time PCR analysis was performed using the precipitated DNA fragments and primers for VWF proximal region, which included the −56 ETS site. (D) Transient transfection of vWF2-Luc or vWF2-ETSm-Luc in HUVECs treated in the absence or presence of TNF-α for 4 hours. The results show the mean + SD of luciferase light units (relative to vWF2 in untreated cells) obtained in triplicate from at least 3 independent experiments. *P < .05. (E) HUVECs were either not transfected (NT) or transfected with control siRNA (CTR) or siRNA against ERG (ERG) and assayed for mRNA expression of ERG or VWF by real-time PCR. The results in E show the mean + SD of mRNA expression (relative to control siRNA-transfected cells) obtained in triplicate from 3 independent experiments. *P < .05, relative to control siRNA. (F) Same HUVECs as described in E were assayed for ERG protein expression using Western blot. For loading control, the blot was stripped and reprobed with antibodies against tubulin and against the nuclear marker, proliferating cell nuclear antigen (PCNA). (G) Parallel plates of HUVECs used for mRNA and protein analysis in panels E and F were transiently transfected with vWF2-Luc or vWF2-ETSm-Luc. The results show the mean + SD of luciferase light units (relative to vWF2 in control siRNA-transfected cells) obtained in triplicate from at least 3 independent experiments. *P < .05; **P < .01; n/s, nonsignificant.

ERG binds to the −56 ETS motif. (A) VWF expression is shown as a function of ERG expression levels. Results are based on the Brown datasets (left) and our compendium of 5532 arrays (right). Red, endothelial cells; black, nonendothelial cells. (B) Quantitative real-time PCR analysis of β-actin, CD31, ERG, friend leukemia virus integration-1, and VWF in HUVECs treated with 20 ng/mL TNF-α for 0 (untreated control), 4, 12, or 24 hours. For each gene, expression is shown relative to control values. Data are expressed as mean + SD of 3 independent experiments. (C) ChIP assay was performed using HUVECs treated in the absence of presence of TNF-α for 12 hours. DNA was sheared, and the resulting DNA-protein complexes were immunoprecipitated in the absence or presence of antibodies to ERG or control IgG. Real-time PCR analysis was performed using the precipitated DNA fragments and primers for VWF proximal region, which included the −56 ETS site. (D) Transient transfection of vWF2-Luc or vWF2-ETSm-Luc in HUVECs treated in the absence or presence of TNF-α for 4 hours. The results show the mean + SD of luciferase light units (relative to vWF2 in untreated cells) obtained in triplicate from at least 3 independent experiments. *P < .05. (E) HUVECs were either not transfected (NT) or transfected with control siRNA (CTR) or siRNA against ERG (ERG) and assayed for mRNA expression of ERG or VWF by real-time PCR. The results in E show the mean + SD of mRNA expression (relative to control siRNA-transfected cells) obtained in triplicate from 3 independent experiments. *P < .05, relative to control siRNA. (F) Same HUVECs as described in E were assayed for ERG protein expression using Western blot. For loading control, the blot was stripped and reprobed with antibodies against tubulin and against the nuclear marker, proliferating cell nuclear antigen (PCNA). (G) Parallel plates of HUVECs used for mRNA and protein analysis in panels E and F were transiently transfected with vWF2-Luc or vWF2-ETSm-Luc. The results show the mean + SD of luciferase light units (relative to vWF2 in control siRNA-transfected cells) obtained in triplicate from at least 3 independent experiments. *P < .05; **P < .01; n/s, nonsignificant.

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