RAD001 in combination with 4-OH tamoxifen or letrozole enhances G
arrest compared with that with monotherapy. (A) Steroid-depleted MCF7-AROM1 and BT474-AROM3 cells were treated for 24 hours with vehicle, androstenedione (10 nM), 4-OH tamoxifen (10 nM), or letrozole (100 nM) alone or in combination with RAD001 (2 nM). Cell cycle was monitored with FACS analysis of cells stained with BrdU and PI. Duplicate plates treated with the drug combination were harvested after 24 hours of treatment. (B) Whole-cell extracts were probed for phosphorylated p27Kip1, cyclin D3, and phosphorylated Rb. Figures below each panel, where shown, represent semiquantitative changes in protein expression relative to actin. (C) The effect of drug treatment on p27Kip1 cellular localization was monitored with confocal microscopy. p27Kip1 was visualized with Alexa 488-conjugated antibodies (green), whereas nuclei were counterstained with TO-PRO-3 (blue). Graphs represent percentage of total nuclei present (represented by DAPI-stained nuclei). Values shown are mean percentages ± standard deviation.