Fig. 6.
ERα Is Required for EMT Induction by E2
A, BG-1 cells were treated with either vehicle (Cont), 10−7 m E2, 10−7/10−8 m DPN, or PPT for 48 h. B, BG-1 cells were transfected with nonspecific siRNA (NS siRNA), ERα siRNA, or ERβ siRNA and subsequently treated with 10−7 m E2 for 48 h. E-cadherin protein (E-Cad) and β-actin were detected by Western blot. C, Cells were transfected with E-cadherin promoter construct and pSV-βGal in combination with the indicated siRNAs. Eight hours after transfection, cells were incubated with vehicle (Cont) or 10−7 m E2 for an additional 48 h. The luciferase activities were calculated relative to the promoterless vector (pGL3-Basic) and expressed as fold change relative to vehicle control. D, Morphological changes of cells were observed by phase-contrast microscope and representative photographs are shown. Original magnification, ×200. E, Scratch was made by scraping the monolayer 24 h after siRNA transfection, and cells were incubated with medium containing vehicle (Cont) or 10−7 m E2. Wound closure was photographed after 24 h. The amount of wound repair was expressed as uncovered area compared with initial uncovered area of vehicle-treated control at time zero. Values are the mean ± sd of three separate experiments. F, SKOV-3 cells were transfected with pCMV5-ERα or without plasmid DNA (mock) for 8 h and subsequently treated with 10−7 m E2 or vehicle (Cont) alone for another 48 h. E-cadherin (E-Cad), Snail, Slug, and β-actin were detected by Western blot. G, Morphological changes of SKOV-3 cells under the same treatment were also observed by phase-contrast microscope, and representative photographs are shown. Original magnification, ×200. The experiments were done in triplicate and repeated thrice. *, P < 0.05 compared with control. Numerical values below each lane of the immunoblots represent quantification of the relative protein level by densitometry (normalized to β-actin protein level). Cont, Control.