The p50 subunit of NF-κB up-regulates Prox1, and both p50 and Prox1 synergistically regulate VEGFR-3 expression. (A) qRT-PCR analysis of Prox1 transcripts in HDLECs_htert treated with IL-3 (10 ng/mL) for 6 hours. (B) qRT-PCR analysis of Prox1 transcripts in RLECs treated with PDTC (2.5μM) for 24 hours. (A-B) Data are presented as β-actin normalized mean transcript expression of 3 independent experiments performed in duplicate ± SEM (total: n = 6 per condition). (C) Prox1 detected by Western blot of nuclear extracts from RLECs treated with PDTC (5μM), MG-132 (250nM), leptomycin B (10nM), or vehicle alone. β-Actin was used as a loading control. Representative data are shown from 1 of 3 experiments. (D) qRT-PCR analysis of Prox1 transcript in H-LLY transfected with p50 and p65 siRNA. Data are presented as the mean transcript expression normalized to β-actin ± SEM derived from 3 independent samples. (E) Prox1-negative nonendothelial line HEK293 was transfected with VEGFR-3^−849/+55 promoter plasmid and pCMV-Flag-p50 or pCMV-Flag-p65 plasmids and cotransfected with pCMV-Prox1 or empty vector (0.25 μg of each plasmid). VEGFR-3 promoter activity was normalized to total milligram of protein. Inset confirms lack of Prox1 in control HEK293 and forced expression in transfected cells. Activation of VEGFR-3 promoter by coexpression of p50 and Prox1 was compared with the effect on NF-κB–independent promoters for phosphoglycerate kinase (PGK) and ubiquitin C (UBC) examined under the same conditions. Data presented as the mean promoter activity ± SEM of 3 independent experiments performed in triplicate (total n = 9 per condition). (F) Prox1-negative blood vascular endothelial line, HULEC, was transfected with VEGFR-3^−849/+55 promoter expression and pCMV-Flag-p50 or pCMV-Flag-p65 plasmids and cotransfected with pCMV-Prox1 or empty vector, as described in panel E. The analysis of the VEGFR-3 promoter activity was performed as described in panel E. Data are presented as the mean VEGFR-3 promoter activity ± SEM derived from 3 independent experiments performed in quadruplicate (total n = 12 per condition). (G) Fold activation of the full-length (−849/+55 bp) and truncated (−118/+55 bp) VEGFR-3 promoters was compared after cotransfection with pCMV-Prox1, pCMV-Flag-p50, and pCMV-Flag-p65 alone or in combination as described in panel E. Data are presented as the mean VEGFR-3 promoter activity ± SEM derived from 3 independent experiments. *P < .05, **P < .01, and ***P < .001 versus control, as determined by Student unpaired t test.