FIGURE 7.

Different modes of action of TPSF and TPBM. A, TPSF does not inhibit binding of E₂-ERα to the flcERE. Fluorescence anisotropy microplate assay was performed as described (19) in the presence of increasing concentrations of TPSF (solid bars) and 5 μm TPBM (hatched bar). Consistent with our detailed dose-response study (19), 5 μm TPBM inhibited binding of TPBM to the flcERE by ∼60%. Data were plotted with the change in anisotropy for binding of E₂-ERα to the flcERE in the absence of small molecule inhibitors (open bar) set to 100% (actual anisotropy: flcERE, 44 mA units; E₂-ERα-flcERE, 81 mA units). Data are the average + S.E. of four experiments. The difference between 5 μm TPSF and the control (no inhibitor) was not significant (p > 0.05). The data for 5 μm TPBM were significantly different from both the control and from 5 μm TPSF (p < 0.01 using Student's t test) B, TPSF decreases ERα levels. MCF-7 cells were cultured in 5% CD calf serum for at least 2 days and maintained in the absence or presence of E₂ and the indicated concentrations of TPSF or TPBM for 24 h and analyzed for ERα by Western blot using 8 μg of protein/lane with actin as internal standard. Data are from the Western blot shown and two additional Western blots from independent experiments and are presented as the mean ± S.E. Quantitation of ERα and actin was by PhosphorImager analysis. The value for ERα/actin in the absence of E₂ was set equal to 1. C, T47D cells were maintained as described under “Experimental Procedures,” maintained in the absence or presence of E₂ and the indicated concentrations of TPSF, harvested, and analyzed by Western blot as described for panel B.