Effect of MIF on HIF-1-dependent gene expressions.

<p>(A) MCF-7 cells were transfected with the indicated plasmids and exposed to 20% or 1% O₂ for 12 h. Then total RNA was isolated. Expression of VEGF, GLUT1, HIF-1α, and 18S rRNA was analyzed by RT-PCR using specific primer pairs. (B, C, D, and E) MCF-7 cells were transfected with pRL-SV40 encoding <i>Renilla</i> luciferase, FLAG-tagged MIF expression vector (B, C, and E), and one of the following plasmids encoding firefly luciferase: HRE reporter p2.1 (<i>B, C, and E</i>) or <i>VEGF</i> promoter reporter pVEGF-KpnI-Luc (<i>D</i>). (F) Constructs encoding the GAL4 DNA-binding domain (amino acids 1–147) fused to the indicated amino acids of HIF-1α were analyzed for their ability to transactivate reporter gene GAL4E1bLuc containing five GAL4-binding sites. MCF-7 cells were co-transfected with pRL-SV40 (50 ng), GAL4E1bLuc (100 ng), GAL4-HIF-1α fusion protein expression plasmids (100 ng), and FLAG-tagged MIF expression vector or empty vector (EV) (200 ng). Cells were treated with 100 µM of DFX or the indicated dose of rhMIF (F) and exposed to 20% or 1% O₂ conditions for 18 h and then harvested. Results shown represent mean±S.D. of three independent transfections. *; <i>p</i><0.05 compared to respective controls without MIF treatment (ANOVA).