Figure 5. Hypoxia mimetics do not induce constitutive luciferase reporter activation.

Equal numbers of Rcho-1 trophoblasts were seeded and incubated for 24 hours. Rcho-1 trophoblasts were then transfected with 0.2 μg pRL-CMV, pRL-SV40, or pRL-TK constitutive luciferase reporter using the split transfection protocol as described earlier. Cells were treated with 100μM Deferoxamine (DFO) or 100μM Cobalt chloride (CoCl₂) or vehicle for 18 hours. (A) Samples were analyzed for constitutive reporter activation. Results are the average of three independent experiments. Luciferase activity at each concentration was analyzed and is shown relative to activity at 21% oxygen. Error bars represent standard deviation. Significance was determined using a one way Anova with Tukeys post hoc test. (B) Nuclear extracts were collected, separated by SDS-PAGE, and Western blotting was performed using a polyclonal HIF-1α antibody and the appropriate secondary antibody as indicated in the Materials and Methods. The same blot was reprobed with actin monoclonal antibody for equal protein loading. Arrows identify HIF-1α actin (45kDa). The figure is a representative of three independent experiments. (C) Densitometric analysis of HIF-1α protein Rcho-1 cells treated with vehicle, CoCl₂ or DFO. Protein levels were normalized to levels of actin.