Effect of MIF-gene silencing on HIF-1 activity.

<p>(A, B, and D) MCF-7 cells were transfected with siRNA against MIF, TRX, CD74, or green fluorescence protein (GFP) as a negative control. Cells were exposed to 20% or 1% O₂ conditions and were harvested for immunoblot assay for MIF or TRX (left panels) or HIF-1α and HIF-1β protein expression (right panels). Representative immunoblots are shown. Intensity of respective bands were analyzed densitometrically and fold induction to lane 1 are plotted accordingly as mean±S.D. (n = 3) (A, B, and C). *<i>p</i><0.05 compared to 20% O₂ conditions without treatment (ANOVA). (C) MCF-7 cells were pre-treated with 10 µM α-tocopherol or 10 mM NAC and then exposed to 20% or 1% O₂ conditions for 4 h. Cells were harvested for immunoblot assay for HIF-1α and HIF-1β protein. (D) siRNAs for GFP or CD74 were introduced into MCF-7 cells. The cells were exposed to 1% O₂ with or without rhMIF treatment. Cells were harvested for immunoblot assay for MIF HIF-1α protein. (E) MCF-7 cells were transfected with HRE reporter p2.1 and pRL-SV40 encoding <i>Renilla</i> luciferase. Cells were treated with the indicated dose of rhMIF 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 (ANOVA).