Inhibition of PAI-1 and induction of PK_LmRNA and protein expression by overexpression of USF-2a under both normoxia and mild hypoxia.

Hepatocytes transfected either with 8 μg USF-2a or ΔHU2a expression vectors or the control vector pCMV were cultured for 24 hours under arterial pO₂. At 24 hours the medium was changed and cells were further cultured for the next 24 hours under normoxic (16% O₂) or hypoxic (8% O₂) conditions. (A, left panels) The PAI-1 and PK_L mRNA levels were measured by Northern blotting (see panel B). The mRNA level under normoxia (16% O₂) was set equal to 100%. (A, right panels) The PAI-1 and PK_L protein levels were measured by Western blotting (see panel B). The protein level under normoxia (16% O₂) was set equal to 100%. The fold induction by 8% PO₂ was calculated in each series to the corresponding 16% values of PAI-1 and PK_L mRNA and protein and is indicated above. (B) Representative Northern and Western blot. For Northern analysis, 15 μg total RNA prepared from the cultured hepatocytes was hybridized to DIG-labeled PAI-1, PK_L, and β-actin antisense RNA probes (see “Materials and methods”). A total of 50 μg of protein from the medium or of the cultured hepatocytes was subjected to Western analysis with an antibody against rat PAI-1 or rat PK_L (see “Materials and methods”). Autoradiographic signals were obtained by chemiluminescence and scanned by videodensitometry. Values are means ± SEM of 3 independent culture experiments. Statistics, Student t test for paired values: *, significant difference 16% O₂ versus 8% O₂; **, significant difference 16% O₂ + USF-2a versus 16% O₂ + pCMV (control); ***, significant difference 8% O₂ + USF-2a versus 8% O₂ + pCMV (control); P < .05. ΔHU2a, USF-2 mutant lacking the second helix of the HLH domain.23