Figure 3

DCQ induces DNA damage and apoptosis via reactive oxygen species. (A) Whole cell lysates of MCF-7 and MDA-MB-231 were prepared after 6 hours of exposure to DCQ (5 μM) under normoxia or hypoxia, and blots were probed for p-H2AX, p53, p-p53, p21, HIF-1α and GAPDH. Results are representative of three independent experiments. (B) MDA-MB-231 and MCF-7 cells were pretreated with 1 mM Vitamin E or DTT for 2 hours followed by 25 min incubation with 10 μM CM-H₂DCFDA dye. Cells were washed with PBS and treated with DCQ for 1 hour under normoxia or hypoxia, after which cells were harvested and the amount of DCF fluorescence was analyzed by flow cytometry. Each percentage is the average ± SE of three independent experiments. (C) MDA-MB-231 and MCF-7 cells were pretreated with 1 mM Vitamin E or DTT for 2 hours followed by 6 hours treatment with DCQ under normoxia or hypoxia. After 24 hours, the extent of DNA fragmentation was determined by TUNEL assay and measured by flow cytometry. One-way ANOVA was used to compare DCQ-treated versus control and statistical significance of p < 0.05 is indicated by *. (D) Whole cell lysates of MCF-7 and MDA-MB-231 were prepared after 6 hours of exposure to DCQ (5 μM) under normoxia or hypoxia, and blots were probed for HIF-1α and GAPDH. Results are representative of three independent experiments.