Activated Notch receptor represses Prox1 expression. (A) Quantitative real-time RT-PCR study shows that adenoviral expression of human NICD in primary human LECs for 48 hours significantly reduced the level of Prox1 mRNA. AdCTR indicates control adenovirus; and AdNICD, NICD-expressing adenovirus. Data are mean plus or minus SD relative to the value of AdCTR. (B) Western blot assay demonstrates a gradual reduction of Prox1 protein level after adenoviral transduction of LECs with NICD at 10, 24, and 48 hours. (C) Western blot assay shows that transient transfection of primary human LECs with a control plasmid vector (CTR, pcDNA3), NICD (pcDNA3-NICD), or Hey1-expressing (pcDNA-hCHF2) plasmids for 48 hours resulted in a significant reduction in the Prox1 protein level. (D) Immunofluorescence analyses of primary LECs that were transduced with either a control (AdCTR, i-iv) or V5-tagged human NICD-expressing (AdNICD, v-viii) adenovirus for 48 hours against nuclei (i,v), Prox1 (ii,vi), V5-tag NICD (iii,vii), and merged images (iv,viii). Arrows in images v to viii point to an LEC with down-regulated Prox1 expression resulting from NICD expression, demonstrating an autonomous regulation of Prox1 by Notch. Bars represent 50 μm. Fluorescent images were acquired using a Zeiss microscope with a 63× objective and processed with AxioVision Digital Imaging software.