Figure 5

DCQ reduced HIF-1α downstream targets. (A, B) ELISA for secreted VEGF in MCF-7 cells and MDA-MB-231 after 12 hours of exposure to DCQ under normoxia or hypoxia. Since 6 hours were insufficient to induce a difference in the secreted VEGF upon exposure to hypoxia, we treated with a lower concentration of DCQ for 12 hours. Results (Average ± SE) are of duplicate measurements of two independent experiments. One-way ANOVA was used to compare DCQ-treated versus control and statistical significance of p < 0.05 is indicated by *. (C) MDA-MB-231 cells were treated with DCQ (5 μM) for 6 hours either under normoxia or under hypoxia. Whole cell lysates were prepared, and blots were probed against TWIST. (D) Trans-well migration assay for cells after 24 hours of exposure to DCQ under normoxia and hypoxia. 2 x 10⁴ MCF-7 cells were seeded on top of matrigel apical insert in serum free media and the lower chamber (the will) was filled with 10% FBS media. Cells were either treated with 2.5 μM DCQ or vehicle (supplemented in the upper chamber only) under normoxia and hypoxia for 24 hours. Then the lower part of the gel was fixed in 4% formaldehyde and cells were stained with Hoechst and counted using fluorescence microscopy. The averages ± SE were obtained from the results of two independent experiments. One-way ANOVA was used to compare DCQ-treated versus control and statistical significance of p < 0.05 is indicated by *.