Fig 6.

Contribution of Pin1 in tamoxifen resistance of breast cancer cells. (A) Reporter gene assays were carried out as described for Fig. 2A in MDA-MB-231 cells transfected with wild-type ERα and vector or Pin1 along with ERE-tk-Luc and CMV–β-Gal. Cells were treated with EtOH or 0.1 μM OHT for 24 h. Relative luciferase levels were determined as the fold difference in luciferase/β-Gal activity relative to that in EtOH-treated vector control samples. (B) GST pulldown assays were performed using extracts from HEK293 cells stably expressing wild-type HA-ERα or HA-ERα S118A mutant that were treated with EtOH, 10 nM E2, or 0.1 μM OHT for 1 h. Extracts were incubated with GST or GST-Pin1, and Western blots (WB) were performed for HA. Input lanes show the presence of equal amounts of HA-ERα and HA ERα S118A proteins in extracts prior to pulldown. (C) Tamoxifen-resistant MCF-7-5C cells were transfected with Pin1 siRNA or control scr siRNA and treated with 1 μM OHT for the indicated length of time. Cell growth was measured using an MTT assay as described in Materials and Methods. Data are shown relative to those of the 0-h time point of scr siRNA-transfected cells. (D) MCF-7 cells overexpressing GFP-Pin1 or GFP were treated with 1 μM OHT for the indicated length of time. Cell growth was assessed spectrophotometrically using crystal violet staining. At harvest, cells were stained with 0.4% crystal violet solution, and following washing, cells were lysed in 50% methanol. Cell growth was quantified by measuring the absorbance at 540 nm of the resultant lysis solution. Data are shown relative to those of the 0-h time point for each cell. Data in all panels are presented as means ± SEM for at least three independent experiments. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.