Concerted regulation of EC-lineage genes by Prox1 and COUP-TFII. (A) FGFR-3 expression is cooperatively activated by Prox1 and COUP-TFII in primary human BECs that were transduced for 48 hours with a control (AdCTR), Prox1 (AdProx1), COUP-TFII (AdCOUP), or both (adBoth) adenovirus based on quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR). (B) A 3-kb proximal promoter construct of mouse FGFR-3 can deliver a Prox1/COUP-TFII–mediated concerted activation when the expression vectors for control, wild-type Prox1 (wt Prox1), DNA-binding defective mutant Prox1 (mutProx1)^6,8  and/or wild-type COUP-TFII (COUP) were transfected into HEK293 with a control empty (pGL2) or FGFR-3 promoter (pGL2-FGFR3) vector.⁶  Activation was determined by luciferase activity after normalization against total protein amount. (C-K) Both Prox1 and COUP-TFII are required to maintain the optimal expression of EC-lineage genes in primary LECs. siRNA duplexes against Prox1 and/or COUP-TFII were transfected in primary LECs for 48 to 72 hours and regulation of EC lineage genes was determined. (C) Protein levels of Prox1, COUP-TFII, FGFR3, VEGFR-3, and β-actin were determined by Western blotting analyses in primary LECs upon knockdown of Prox1 and/or COUP-TFII for 72 hours. Messenger RNA levels of FGFR-3 (D), VEGFR-3 (E), LYVE-1 (F), neuropilin-1 (NP-1, G), podoplanin (H), ICAM-1 (I), and MCP-1 (J) were determined in primary LECs, of which Prox1 and/or COUP-TFII were knocked down for 48 hours () or 72 hours (■) by quantitative real-time RT-PCR. All data are expressed as an average (SD, *P < .05) compared with the corresponding control siRNA samples. (K) Messenger RNA levels of Prox1, COUP-TFII, ABCA4, versican, and β-actin were determined by semiquantitative conventional RT-PCR analyses in primary LECs after knockdown of Prox1 and/or COUP-TFII for 72 hours.