Figure 2: ER^muts confer antiestrogen resistance when expressed alone.

SKBR3 (ER- negative breast cancer) cells were plated in phenol red free media and transfected with an estrogen responsive reporter gene (3X-ERE-tata-Luc) in the presence of ER^WT, ER^Y537S or ER^D538G. After 5 hours, cells were treated with 17β-estradiol (1 nM) (A) and ER antagonists (10⁻¹² M to 10⁻⁶ M) (B-I). Firefly luciferase activity was assessed and normalized to β-galactosidase transfection control (Y-Axis). Data points are the mean of three technical replicates, and error bars are the standard deviation of these replicates. Data presented is a representative of three independent experiments. Two-way ANOVA was utilized, comparing the logIC₅₀s of all three independent experiments, to determine if there were significant differences between the WT and mutant receptors. Significant differences (p-value < 0.05) of the mutant IC₅₀s when compared to that of the WT that were determined by this analysis are represented with a star. For GDC-0810 and AZD9496 on ER^Y537S the highest dose tested (10⁻⁶ M) was used as a surrogate, as the IC₅₀ is greater than this value. The only compound that did not reach a significant difference for either mutant isoform was lasofoxifene.