Figure 4.

PELP1 Modulation of Aromatase Requires Nongenomic Signaling

A, MCF7 cells were transiently transfected either with ERα siRNA or control siRNA. After 72 h, cells were transfected with Aro-1.3/II-Luc, β-Gal reporter gene along with or without PELP1, and 48 h later, reporter gene activation was measured. Inset shows Western analysis of ERα in control and siRNA-transfected cells. B, MDA-MB-231 cells were cotransfected with the Aro-1.3/II-Luc reporter with or without PELP1 (left panel) and with or without PELP1 shRNA (right panel). After 48 h, cells were stimulated with or without EGF (100 ng/ml) for 12 h, and luciferase activity was measured. C, MCF7 cells were cotransfected with Aro-1.3/II luciferase reporter with or without PELP1. After 48 h, cells were pretreated for 1 h with p42/44MAPK inhibitor (PD98059, 20 μm), p38MAPK inhibitor (SB203580, 40 μm), Src inhibitor PP2 (10 μm), or PI3K inhibitor (LY294002, 20 μm) and then stimulated with EGF for 12 h. Luciferase activity was measured. D, Src-negative SYF cells were transiently cotransfected with Aro-1.3/II along with or without PELP1 and c-Src expression vectors. After 48 h, luciferase activity was measured. E, MCF7-HER2 cells were cotransfected with an Aro-1.3/II luciferase reporter and β-Gal reporter gene along with PELP1. After serum starvation for 48 h, cells were treated with or without 10 μm Src inhibitor PP2 for 12 h. Luciferase activity was measured. In all reporter gene assays, parental vectors were used as controls, and the total amount of the DNA in the transfection was kept constant by adding appropriate empty vectors. β-Gal values were used to normalize luciferase activity for transfection efficiency. *, P < 0.05; **, P < 0.001. Data shown are the means ± se from three independent experiments performed in triplicate wells.