<p>(A and B) MCF-7 cells were transfected with 2 µg of p3xFLAG-CMV-10 plasmid (EV) or pFLAG-MIF plasmids (A and B lane 4; 2 µg, B lane 3; 0.5 µg). (C) MCF-7 cells were transfected by the plasmids for expression of FLAG tag (EV) and FLAG-tagged MIF and selected by puromycin resistance. Two clones of MCF-7 cells stably expressing FLAG tag (EV) and FLAG-tagged MIF (MIF#1 and MIF#2 cells) were established. (D) MCF-7 cells were treated with the indicated doses of bacterially produced recombinant human (rh)MIF. (E) MCF-7 cells were treated with rhMIF. Cells were exposed to 20% (A, B, C, and D), 5% (C), or 1% (A, B, C, and D) O<sub>2</sub> conditions or treated with 100 µM DFX (E) for 4 h. Cell were harvested for immunoblot assay for MIF (A and C), HIF-1α and HIF-1β protein (A, B, C, D, and E). Representative immunoblots are shown. Intensity of respective bands were analyzed densitometrically and fold induction to respective controls (lane1) are plotted accordingly as mean±S.D. (n = 3) *<i>p</i><0.05 compared to 20% O<sub>2</sub> conditions without MIF (lane 1) (ANOVA).</p
