OxPLs do not influence VEGF mRNA stability and do not up-regulate HIF-1α protein or HIF-1–dependent transcription. (A) HUVECs were incubated in medium 199 containing 2% FCS with or without 130 μM OxPAPC. Actinomycin D was added after 4 hours to stop transcription. After indicated time intervals, the samples were collected and RNA was extracted, reverse transcribed, and analyzed by RT-qPCR for expression of VEGF or COX-2 mRNA. (B) HUVECs were incubated in medium 199 containing 2% FCS with or without 130 μM OxPAPC or 30 mM metal chelator Tiron. At the indicated time points, the cells were scraped into Laemmli sample buffer and analyzed by Western blotting. Staining of the same samples for HIF-1α or EGR-1 proteins is shown. (C) HUVECs grown in 6-well dishes were cotransfected with firefly luciferase promoter-reporter driven by hypoxia response elements and constitutively active Renilla luciferase. After 24 hours, the medium was changed to 2% FCS containing indicated concentrations of OxPAPC (100 μg/mL of OxPAPC = 130 μmol/L). After an overnight incubation, the cells were scraped and activities of both luciferases measured using a dual luciferase assay system. The data represent a ratio of firefly luciferase activity to that of Renilla luciferase. Error bars represent SD.