Figure 6

EGFR signaling is not required for the autocrine regulation of cell proliferation by ERα. A, MCF-7 cells treated with 10 nM ICI182780 for 72–96 hr, showing the down-regulation of ERα and Ki-67 by ICI182780 treatment. In A and B, ERα and Ki-67 were evaluated by immunofluorescent staining, nuclei were stained with DAPI. ERα-staining and Ki-67 staining were overlayed to show colocalization (Merge). Magnification, 400×. B, EGFR inhibitor Gefitinib does not inhibit E2 induced ERα and Ki-67 expression. MCF-7 cells were stimulated with 100 nM E2 in the presence of 10 nM ICI182780 and 20 μM EGFR inhibitor Gefitinib. MCF-7 cells were treated first with 10 nM ICI182780 for 72–96 hr, followed by adding the EGFR inhibitor Gefitinib to a final concentration of 20 μM for 2 hr, before adding estrogen to a final concentration of 100 nM for 16 hr. C, flow cytometry analysis of cell cycle progression, showing E2 stimulated cell cycle progression is not inhibited by Gefitinib. Upper panel, MCF-7 cells treated with ICI182780 for 72 hr. The cells were arrested at G1 phase. Lower panel, cell cycle progression was stimulated by 100 nM E2 in the presence of 10 nM ICI182780 and 20 μM EGFR inhibitor Gefitinib. MCF-7 cells were treated first with 10 nM ICI182780 for 72 hr, followed by treatment with the Gefitinib (20 μM) for 2 hr, before stimulation with 100 nM E2 for 36 hr. D, immunoblotting of phosphorylated ERK1/2, showing the inhibition of EGFR signaling by 20 μM Gefitinib. MCF-7 cells were serum starved for 24 hr, followed by treatment with 20 μM Gefitinib for 2 hr, before EGF stimulation for 5 min. The control group of cells was not treated with Gefitinib. Phosphorylation of ERK1/2 was used to assess the activation of the EGFR signaling.