Figure 6. EHT 1864 inhibits activity of the promoter of the ER gene but does not disrupt ER stability.

(A) T47D cells were deprived of estrogen for 24 hours and then transfected with a WT ER gene promoter-luciferase or the ER gene promoter lacking the enhancer (delta enhancer) reporter plasmid. Cells were treated with either vehicle or 1 nM estradiol in the presence or absence of 10 μM EHT 1864. Luciferase activity was determined 48 hours after transfection and normalized for protein. Results are reported as values relative to ER-luciferase WT set at 100%. Data represent the mean of three experiments performed in triplicate ± SEM. (B) T47D cells were deprived of estradiol for 24 hours and then treated with vehicle or 10 μM EHT 1864 for 24 hours. ER protein levels were determined by western blot and actin was used as a loading control. (C) T47D cells were deprived of estradiol for 24 hours and then pretreated with vehicle or 10 μM EHT 1864 for 1 hour followed by addition of cycloheximide. Cells were harvested and protein lysates obtained at the indicated times after cycloheximide treatment. ER protein levels were determined by western blot and actin was used as a loading control. This is a representative experiment of two experiments. P-values were determined by student t-test. *P-value < 0.05.