Figure 4.

(A) ERβ1 knockdown experiments in DU-145 and MDA-MB-231 cells. Cells were transfected with 50 nM siRNA against ERβ1 or scrambled siRNA (siCONTROL), as described in the Materials and Methods section. After 72 hours of incubation, total RNA was extracted and semiquantitative RT-PCR was performed using specific primers against ERβ1 and β-actin. Similar results were obtained from two separate experiments. (B–E) Antiproliferation effects of apigenin or genistein on ERβ1 knockdown DU-145 and MDA-MB-231 cells. siRNA against ERβ (siESR2)- and siRNA scrambled control (siCONTROL)-transfected cells were incubated with 10 or 20 µM apigenin (B and C) or genistein (D and E) for 72 hours and subsequently analyzed for cytotoxicity by MTS assay, as described in the Materials and Methods section. Control groups (Lipofectamine2000) without siRNA transfection were treated with vehicle (CTL). The viability of the cells with apigenin or genistein treatment (siESR2 + phytoestrogen or siCTL + phytoestrogen) was normalized to values obtained in cultures treated similarly but without phytoestrogens (siESR2 alone and siCTL alone). The cell viability in each treatment group was calculated as a percentage of the value found in untreated controls without siRNA transfection. Data represent the average of three separate experiments, with standard deviation indicated. ^aThe mean of the treated group was statistically different from that of untreated controls at P < .05. ^bNo significant difference between the treated group and the untreated control.