Hypoxia-dependent induction of PAI-1 mRNA and protein expression in primary rat hepatocyte cultures. Hepatocytes were cultured for 24 hours under arterial pO2. At 24 hours, the medium was changed and cells were further cultured for the times indicated under normoxic (16% O2) and hypoxic (8% O2) conditions. (A) In each experiment, the PAI-1 mRNA level measured by Northern blotting (cf B) at 0 hour under normoxia (16% O2) was set equal to 100%. The PAI-1 protein level measured by Western blotting (cf B) at 0 hour under normoxia (16% O2) was set equal to 1%. Values are means ± standard error of mean (SEM) of 3 independent culture experiments. Statistics, Student's t-test for paired values: * significant differences 16% O2 versus 8% O2,P ≤ .05. (B) Representative Northern and Western blot. For Northern analysis, 15 μg total RNA prepared from the cultured hepatocytes were hybridized to DIG-labelled PAI-1 and β-actin antisense RNA probes (cf, Materials and Methods). A total of 50 μg of protein from the medium of the cultured hepatocytes was subjected to Western analysis with an antibody against rat PAI-1 (cf, Materials and Methods). Autoradiographic signals were obtained by chemiluminescence and scanned by videodensitometry.
