Figure 3

EGFR promotes EMT and its down-regulation is involved in ERβ1-induced E-cadherin expression. (A) EGFR, total ERK1/2 and phospho-ERK1/2 levels in control (Lenti), ERα- and ERβ1-expressing MDA-MB-231 cells following incubation with or without E2 for 24 h. (B) EGFR protein levels in control (Lenti) and ERβ1-expressing Hs578T cells. (C) EGFR protein levels in MDA-MB-231 cells transiently transfected with control or ERβ siRNA (3#). (D) ERβ1-expressing MDA-MB-231 cells were incubated in absence or presence of 5 ng/ml TGF-β1 or 10 ng/ml EGF for 24 h and photographed (scale bars, 50 μm). (E) ERβ1-expressing MDA-MB-231 cells were incubated in absence or presence of 10 ng/ml EGF for 24 h and analyzed for the expression of EGFR, total ERK1/2 and phospho-ERK1/2 by immunoblotting. Note that the decreased EGFR levels following EGF treatment is due to increased degradation. (F) miR-200a, miR-200b and miR-429 levels in control (Lenti) and ERβ1-expressing MDA-MB-231 cells following incubation with 5 ng/ml TGF-β1 or 10 ng/ml EGF for 24 h. The graph shows the data as fold change compared with the untreated Lenti cells (mean of three separate experiments (± SEM) with P-value (*) ≤0.05%). (G) E-cadherin mRNA and protein levels in ERβ1-expressing MDA-MB-231 cells following incubation with 5 ng/ml TGF-β1 or 10 ng/ml EGF for 24 h. The graph shows the mean of three separate experiments with SEM and P-value (*) ≤0.05% indicated. (H) ERβ1-expressing MDA-MB-231 cells were stably co-transfected with an empty pBABE vector (ERβ1:pBABE cells) or the pBABE-EGFR plasmid (ERβ1:EGFR cells), photographed and analyzed for EGFR, E-cadherin and ERβ1 expression by immunoblotting (scale bars, 50 μm).