![Fig. 3. Inhibition of PAI-1 and induction of PKLpromoter–controlled Luc expression by overexpression of USF-2a under both normoxia and mild hypoxia. / (A) The 5′-flanking region of the rat PAI-1 gene with its footprinted sites [A-G]. In the “C-site” the 2 potential USF-2a binding elements (HRE) matching the E-box consensus sequence are underlined. S (G/C) is shown because the actual PAI-1 sequence does not match bases allowed by the consensus. (B) Hepatocytes were transiently cotransfected with the pGl3PAI-766 Luc or pGl3PKL-183 Luc constructs and the USF-2a, ΔHU2a, or ΔTDU2 expression vectors or the control pCMV vector. After 24 hours the transfected cells were cultured for the next 24 hours under hypoxic (8% O2) or normoxic (16% O2) conditions, as indicated. In each experiment the percentage of Luc activity was determined relative to the normoxic pGl3PAI-766 Luc or pGl3PKL-183 Luc controls, which were set equal to 100%. The fold induction by 8% O2was calculated in each set of transfections relative to the corresponding Luc activity obtained under 16% O2. 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 or ΔTDU2 versus 16% O2 + pCMV (control); ***, significant difference 8% O2 + USF-2a or ΔTDU2 versus 8% O2 + pCMV (control); P < .05. ΔHU2a, USF-2 mutant lacking the second helix of the HLH domain; ΔTDU2, USF-2 mutant lacking the first 198 amino acids of the transactivation domain.23 L1-L4: L indicates liver-specific; L1, binding site for hepatocyte nuclear factor 1, L2, binding site for nuclear factor 1; L3, binding site for hepatocyte nuclear factor 4; L4, binding site for USF.3132](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/97/9/10.1182_blood.v97.9.2657/6/h80910984003.jpeg?Expires=1724259984&Signature=Jba8DIC5dRzE35V44EOPOvGlLaBgoQNU9Z-baAgGCo8clPVCRC40khK~ESICmUNGBBbeO9TxiciPgbZM3WY-JMHZpQgX~WnlEABK4mHVP6ZVTITMzfAUC5R~pJpWH9gKtF5KkcvclbNg4yPZhghKDVKUNZPc742FnDIoMMLNIwBTQpwnhDw7CUwDH8mNJsvyF6xCV-QGFag~i2~DEVoslLPUVXlw1~rvwnUKTuB0XHtcCofuLetqHGjb2lFoGY6qM4m1c~neksFsLGDsrQ3qFqDUb-oD~XqL5~QlmK-ZdzTNyD7KueF552jYtTs2QT09RlxP1WKeMtHpJDvcdYYq6g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Inhibition of PAI-1 and induction of PKLpromoter–controlled Luc expression by overexpression of USF-2a under both normoxia and mild hypoxia.
(A) The 5′-flanking region of the rat PAI-1 gene with its footprinted sites [A-G]. In the “C-site” the 2 potential USF-2a binding elements (HRE) matching the E-box consensus sequence are underlined. S (G/C) is shown because the actual PAI-1 sequence does not match bases allowed by the consensus. (B) Hepatocytes were transiently cotransfected with the pGl3PAI-766 Luc or pGl3PKL-183 Luc constructs and the USF-2a, ΔHU2a, or ΔTDU2 expression vectors or the control pCMV vector. After 24 hours the transfected cells were cultured for the next 24 hours under hypoxic (8% O2) or normoxic (16% O2) conditions, as indicated. In each experiment the percentage of Luc activity was determined relative to the normoxic pGl3PAI-766 Luc or pGl3PKL-183 Luc controls, which were set equal to 100%. The fold induction by 8% O2was calculated in each set of transfections relative to the corresponding Luc activity obtained under 16% O2. 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 or ΔTDU2 versus 16% O2 + pCMV (control); ***, significant difference 8% O2 + USF-2a or ΔTDU2 versus 8% O2 + pCMV (control); P < .05. ΔHU2a, USF-2 mutant lacking the second helix of the HLH domain; ΔTDU2, USF-2 mutant lacking the first 198 amino acids of the transactivation domain.23 L1-L4: L indicates liver-specific; L1, binding site for hepatocyte nuclear factor 1, L2, binding site for nuclear factor 1; L3, binding site for hepatocyte nuclear factor 4; L4, binding site for USF.31 32
