Kinetics of hypoxia-induced PAI-1 mRNA and protein.

HepG2 cells were cultured at oxygen concentrations of 1%, 2%, 3.5%, 5%, 6.5%, 8%, and 21% for up to 48 hours. At the indicated intervals, RNA, protein, and medium were harvested. (A) Levels of PAI-1 mRNA were determined by real-time RT-PCR. The levels of PAI-1 mRNA were normalized to 18S rRNA within each sample and to the starting values, which were set to 1.0. Each graph represents the average of 2 separate experiments, each analyzed in duplicate on 2 separate occasions (n = 8). Error bars represent SEM; *P < .001 versus respective ambient controls. (B) For each oxygen tension one representative Western blot of PAI-1 is shown. Autoradiographic signals were obtained by chemiluminescence and detected in a multi-imager. (C) Quantitative measurements of the PAI-1 protein detected by the Western blots. The signals were normalized to the total amount of protein transferred to the membranes. Each of the graphs represents the average of 2 separate experiments, each analyzed in duplicate (n = 4). Error bars represent SEM; *P < .005 versus respective ambient controls. (D) Determination of soluble PAI-1 protein in the cell culture medium was performed by enzyme-linked immunosorbent assay. Each of the graphs represents values from 2 separate experiments, each determined in triplicate (n = 6). Error bars represent SEM; *P < .001 versus ambient controls.