Functional role of HAF in hypoxia-induced expression of the epo gene.

(A) Northern analysis of Hep3B clones expressing HAF-antisense RNA. Individual clones (1-8) stably transfected with either HAF cDNA in reverse orientation (lanes 1-7) or the vector alone (lane 8); lanes 9 and 10 represent controls with non-transfected Hep3B cells and NN10 cells. Total RNA was isolated, size fractionated and hybridized with the HAF cDNA probe. The thin arrow indicates the 2.6-kb HAF transcript. The thick arrow indicates the presence of the 0.7-kb HAF antisense-RNA in clones 2, 4, 5, and 6 but not in wild-type (clone 9) or clones transfected with vector alone (8). The bottom panel shows β-actin mRNA. (B) Effect of HAF-antisense-RNA on hypoxic induction of epo mRNA. Two clones (4 and 5 of panel A) expressing HAF antisense RNA (lanes 3-6), and a control clone (8 of panel A) (lanes 1 and 2) transfected with vector alone were exposed to hypoxia (2% O₂) for 48 hours (lanes 2, 4, and 6), or under normal oxygenation (lanes 1, 3, and 5). The probe used was a monkey epo cDNA. The arrow indicates 1.4-kb epo mRNA. (C) Effect of HAF antisense RNA on hypoxia induced expression of VEGF mRNA. The membrane in panel B was stripped of the epo probe and hybridized to human VEGF cDNA. Arrows indicate different transcripts of VEGF. (D) The same membrane was stripped and rehybridized with mouse β-actin cDNA to control for loading differences.