Figure 4

Dependence of phytochemical-induced expression of BRCA2 on BRCA1. (A) BRCA1-siRNA causes time-dependent loss of BRCA1 and BRCA2 in MCF-7 cells. Subconfluent proliferating cells were treated with BRCA1-siRNA (left) or control-siRNA (right) (50 nM) for different times, harvested, and Western blotted for BRCA1, BRCA2, and actin. (B) Effect of BRCA1-siRNA on BRCA2 protein levels and vice versa in DU-145 cells. Cells were treated with BRCA1, BRCA2, or control-siRNA (50 nM) for 72 h and Western blotted for BRCA1, BRCA2, and actin. Results are shown for two separate cell treatments and protein isolations on the same blot. (C) Effect of wtBRCA1 on BRCA2 protein levels and vice versa in DU-145 cells. Cells were transfected overnight with wtBRCA1, wtBRCA2, or empty pcDNA3 vector, washed, postincubated for 24 h to allow gene expression, harvested, and Western blotted for BRCA1, BRCA2, and actin. Results are shown for two separate cell treatments and protein isolations on the same blot. The densitometry values are means±ranges of two experiments. (D) Effect of BRCA1 and BRCA2 siRNAs on BRCA induction by I3C. DU-145 cells were preincubated with the indicated siRNA (50 nM × 72 h) or no siRNA (transfection reagent only), then treated with I3C (40 μM) for 24 h, and then harvested for Western blotting. (E) Effect of BRCA1 and BRCA2 siRNA on induction of BRCA1 and BRCA2 by genistein. DU-145 cells were preincubated with the indicated siRNA (50 nM × 72 h), treated with genistein (5 μM) for 24 h, and harvested for Western blotting as above. (F) Induction of BRCA1 and BRCA2 by a combination of I3C plus genistein. MCF-7 or DU-145 cells were treated with low doses of I3C (25 μM) and/or genistein (1 μM) for 24 h and harvested for Western blotting. The densitometry values are means±ranges of two independent experiments. (G) Effect of ICI182,780 on phytochemical induction of BRCA1 in MCF-7 cells. Proliferating cells were incubated with the indicated agents for 24 h and then harvested for Western blotting for BRCA1, ER-α, or actin. (H) Effect of BRCA1 knockdown and phytochemicals on ER-α protein levels. MCF-7 cells were pretreated with BRCA1 or control siRNA as described above, exposed to the indicated doses of I3C or genistein for 24 h, and then Western blotted for ER-α, BRCA1, or actin.