Figure 4. HDACs regulate AKT mRNA expression by an estrogen receptor-(α) dependent mechanism.

(A) MCF7 cells were treated with vehicle (V), 0.1 µM PCI-24781 (P), 10 µM OH-tamoxifen (T), or the combination (PT) for 72 hours and western blotted. (B) MCF7 cells were treated with 0.1 µM PCI-24781 and ESR1 mRNA levels were measured at the indicated times and presented relative to untreated ESR1 levels. (C) The indicated cell lines were treated with vehicle (V) or 0.1 µM PCI-24781 (P) for 6 hours and ESR1 mRNA levels were measured and presented relative to vehicle treated MCF7 cells. (D) MCF7 cells were treated with vehicle (Veh), 0.1 µM PCI-24781 (PCI), 10 µM OH-tamoxifen (Tam), 0.1 µM PCI-24781 and 10 µM OH-tamoxifen (PCI+Tam), 0.1 µM fulvestrant (Ful), or 0.1 µM PCI-24781 and 0.1 µM fulvestrant (PCI-Ful) for 24 hours and AKT1 mRNA levels were measured and presented relative to vehicle treated MCF7 cells. (E) MCF7 cells were transfected with scramble or increasing concentrations ESR1 directed siRNA for 72 hours and assayed for AKT1 and AKT2 mRNA and western blotted for ER and Akt pathway components. AKT1 and AKT2 expression are normalized to individual scramble treatments and not to each other. For both AKT1 and 2, all ESR1 siRNA concentrations resulted in significant reductions (P-value < 0.05) compared to scramble transfection. (F) MCF7 cells were transfected with scramble or 1 µM ESR1 directed siRNA for 24 hours, divided and then treated with vehicle (V) or 0.1 µM PCI-24781 (P) for 72 hours and evaluated by western blot. For all mRNA measurements, experiments were conducted in triplicate and results expressed as the average with the error bars indicating the standard error of the mean. An (*) indicates a significant difference (P-value < 0.05) and a (#) an insignificant difference (P-value > 0.05) compared to vehicle or zero time treatment. A (@) indicates a significant (P-value < 0.05) difference compared to PCI-24781 treatment.