Figure 2.

Effect of CoCl₂ and E₂ on HIF-1α levels in ECC-1 cells. A, Dose response to CoCl₂ on HIF-1α protein. Cells were treated with 0 (vehicle), 1, 10, or 100 μm CoCl₂ for 4 h, and HIF-1α and total Akt protein levels were examined by Western blot analysis (30 μg protein/lane). B, Effect of CoCl₂ over time on HIF-1α protein. Cells were treated with 100 μm CoCl₂ for 0–72 h, and HIF-1α and HIF-1β protein levels were examined by Western blot analysis (30 μg of protein/lane). C, Effect of E₂ plus CoCl₂. Cells were treated with vehicle, 10 nm E₂, 100 μm CoCl₂, or the combination of the two for 2 or 4 h, and HIF-1α and HIF-1β protein levels were examined by Western blot analysis (40 μg protein/lane). D, Cells were treated as described in C and HIF-1α and 18S mRNA levels measured by RT-PCR. Real-time RT-PCR results (mean ± sem, n = 6 replicate cultures/group; *, P < 0.001 vs. vehicle, 2 h CoCl₂, 2 h E₂, 2 h CoCl₂ + E₂ and 4 h CoCl₂; **, P < 0.01 vs. vehicle, 2 h CoCl₂, 2 h E₂, and 4 h CoCl₂). E and F, Cytoplasmic and nuclear localization of HIF-1α protein induced by CoCl₂, E₂, or CoCl₂ plus E₂ (concentrations as in C) for 4 h. HIF-1α and Akt protein levels were examined in nuclear and cytoplasmic fractions by Western blot analysis. E, Representative gels (10 μg protein/lane). F, Quantification of HIF-1α protein by densitometry after normalization to Akt (fold increase compared with vehicle; means ± sem, n = 5 replicate cultures/group; *, P < 0.01, **, P < 0.0001, #, P < 0.05, ##, P < 0.0001 vs. all other groups).