Fig. 4

NRF1 bound to the Ace and Edn1 promoter regions and activated gene expression. A HUVECs were cultured in 1% O₂ pre-equilibrated medium for the indicated duration at 37 °C under 1% O₂. To identify the interaction between NRF1 and the Edn1 or Ace promoter, cells were fixed and lysed for ChIP experiments. Enriched DNA fragments were amplified by real-time PCR (mean ± SD, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by Student’s t test). B–D A luciferase reporter gene assay was carried out after cotransfection of an ACE (−348/+20 Luc) or EDN1 (−221/+63 Luc) plasmid and the pRL-TK vector to normalize the transfection efficiencies. To interfere with NRF1 levels, HEK 293T cells were cotransfected with NRF1 overexpression plasmids (NRF1 OE, B) under normoxia or with NRF1 siRNA (siNRF1) under normoxia (C) or hypoxia (D). The results were normalized with the luciferase/Renilla ratio (mean ± SD, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by Student’s t test). Nor normoxia, Hyp hypoxia