PROX1 upregulates HEY1 in primary LECs.

<p>(A) Knockdown of PROX1, but not COUP-TFII, resulted in downregulation of HEY1 in LECs. qRT-PCR analyses were performed to determine the expression of HEY1, PROX1 and COUP-TFII in primary LECs that were transfected for 48 hours with siRNAs targeting the firefly luciferase (siCTR), PROX1 (siProx1), COUP-TFII (siCOUP) or both PROX1 and COUP-TFII (siBOTH). (B) Adenoviral expression of PROX1, but not of COUP-TFII, increased the expression of HEY1 mRNA in LECs. qRT-PCR analyses were performed to determine the level of HEY1 mRNA in LECs that were transduced with a control (AdCTR), PROX1 (AdProx1), COUP-TFII (AdCOUP) adenovirus or with both PROX1 and COUP-TFII (AdBOTH) for 48 hours. (C) Luciferase reporter constructs of the promoters of HEY1, Hey2 or HeyL were transiently transfected into HEK293 cells along with a control vector (Empty Vector), PROX1 wild type (Prox1_WT) or PROX1 DNA-binding mutant (Prox1_Mut). After 48 hours, luciferase activity was measured and each value was normalized by total cell lysate amounts. (D) PROX1 also upregulated HEY1 in aortic endothelial cells (human umbilical aortic endothelial cells, HUAEC). Relative expression was determined for HEY1, Hey2 and PROX1 in primary HUAECs that were transduced with a control or PROX1-adenovirus for 48 hours. (E) Chromatin immunoprecipitation (ChIP) assay demonstrating a physical association between PROX1 protein and the HEY1 promoter in LECs. Primary LECs were transfected with a control (LEC/CTR) or a PROX1-expressing vector (LEC/Prox1) for 48 hours and then subjected to ChIP analyses using a normal IgG (IgG) or anti-PROX1 (α-Prox1) antibodies. (F) A ∼0.6-kb HEY1 proximal promoter was sufficient to deliver the PROX1-mediated repression of HEY1 expression. Reporter constructs of the HEY1 promoter were transfected with a control or PROX1-expressing vector into HEK293 cells for 48 hours. (G) Ectopic expression of PROX1 in LECs resulted in downregulation of endogenous PROX1. LECs were transduced with a control (AdCTR) or PROX1 (AdProx1) adenovirus. After 48 hours, qRT-PCR was performed by using two sets of probes that target the 3′ untranslated region (UTR) of endogenous PROX1. All data were shown as a relative average expression ± standard deviation (SD).