Hypoxia stimulates transcription from 3TP-Lux in HUVECs.
(A) Structural organization of the 3TP-Lux reporter plasmid, composed of 3 AP-1 binding sites and a TGF-β–responsive PAI-1 promoter fragment driving a luciferase reporter gene, is shown. (B) Response of 3TP-Lux is increased by 4-fold in HUVECs and 10-fold in HepG2 cells after exposure to 1% O2 or TGF-β2 (12.5 ng/mL) for 24 hours. Luciferase activity was determined and results corrected to CMV promoter-derived β-gal activity and protein content of the extracts. Results from 3 independent experiments are shown. (C) Response of p3TP-Lux to hypoxia or TGF-β2 (12.5 ng/mL) is absent in the Smad4-defective MDA-MB-468 cell line and can be reinstated by Smad4 expression. MDA-MB-468 in parallel with the lung carcinoma cell line A549 that expresses Smad4 was transfected with p3TP-Lux by electroporation either in the presence of Smad4 expression vector or empty vector. Transfected cells were divided into 3 identical culture dishes and were exposed to 20% O2 with or without TGF-β2 (12.5 ng/mL) or were exposed to 1% O2 for 24 hours. Luciferase activity from 3 independent experiments is shown.
