Prox1 directly activates the VEGFR-3 promoter. VEGFR-3−849/+55 promoter plasmid was cotransfected with pCMV-Prox1 plasmid (0-0.5 μg) in H-LLY cells (A) and RLECs (B). Promoter activity was measured by luciferase assay and normalized per milligram of protein. Note linear response to Prox1 transactivation in both cell lines as determined by linear regression (r2 shown on graph) of the mean promoter activity ± SEM of 3 independent experiments performed in duplicate (n = 6 per condition; A-B). (A-B) *P < .05 versus control, **P < .01 versus control, ***P < .001 versus control, by Student unpaired t test. (C) ChIP analysis of the VEGFR-3 promoter was performed on RLECs as described in the legend for Figure 4. Immunoprecipitated chromatin was visualized by PCR with primers flanking transcription factor binding sites (−403/−238 bp) or upstream of binding sites (−813/−403 bp). Data are representative of 3 independent ChIP experiments with similar results. (D) Fold activation of a truncated VEGFR-3 promoter (−436/−254) was compared with the full-length VEGFR-3−849/+55. RLECs were cotransfected with 0.5 μg of VEGFR-3−849/+55 or VEGFR-3−436/−254 promoter plasmids and 0.5 μg of pCMV-Prox1, pCMV-Flag-p50, pCMV-Flag-p65, or empty control plasmid. Promoter activity is normalized per milligram of protein. Data are presented as the mean promoter activity of 3 independent experiments performed in duplicate ± SEM (total n = 6 per experimental condition). ns denotes nonsignificant changes. **P < .01 versus control as determined by Student unpaired t test.
