Regulation of Prox1 and COUP-TFII by Notch ligands. (A) Notch ligands, Jag1 and Dll4, down-regulate Prox1 and COUP-TFII in primary LECs. Soluble Jag1 or Dll4 recombinant protein was either added to culture media (200 ng/mL) of LECs or precoated (1 μg/mL) on culture dishes before LEC culturing. After 24-hour activation by the soluble ligands in media (M) or 48-hour activation by precoated ligands (C), LECs were harvested for quantification of the expression of Prox1 and COUP-TFII. (B-C) Early response in the expression of Hey1, Prox1, and COUP-TFII in LECs by soluble Notch ligands. Soluble Jag1 (100 ng/mL; B) or Dll4 (100 ng/mL; C) was added in LEC-culture media, and total RNA was harvested after 0, 30, 60, and 120 minutes for quantitative RT-PCR analyses. (D) Regulation of Hey1, Prox1, and COUP-TFII in LECs on inhibition of Notch signal pathway. LECs were treated with dimethyl sulfoxide (DMSO; vehicle) or Notch inhibitor DAPT (60 nM), and total RNA was harvested after 24 hours for quantitative RT-PCR analyses. Data are mean ± SD relative to the value of phosphate-buffered saline (PBS) treatment (A), 0-hour time point (B-C), or DMSO treatment (D).