Effects of RAD001 on ER-mediated transcription. (A, B) Cell lines, co-transfected with EREIItkLuc and pCH110, were treated with RAD001 (2 nM) alone (basal ER-mediated transcription) or, to represent E2-mediated transcription, with a standard 10 nM concentration of androstenedione ± 4-OH tamoxifen (10 nM) or letrozole (100 nM) ± RAD001 (2 nM). (C) LTED cells were treated similarly, except that E2 (0.01 nM) was used in place of androstenedione. Luciferase activity was normalized by β-galactosidase from co-transfected pCH110. Normalized luciferase activity from triplicate wells was expressed relative to the vehicle-treated control. Bars represent ± SEM. *P < 0.05, derived from the comparison of vehicle (DCC) versus RAD001 for basal transcription or the endocrine agent alone versus the combination with RAD001 with Student unpaired t test. Effects were confirmed in two independent experiments. (D) LTED cells were treated as shown for 24 hours. Western blot was used to assess changes in phosphorylation of the ER in response to RAD001. Figures below each panel, where shown, represent semiquantitative changes in protein expression relative to actin.