Figure 4.

Mutations of NES-ERα impair nuclear export of full-length ERα and prevent S phase entry in MCF-7 cells stimulated by estradiol. (a) The wild-type NES-ERα endowed in the amino acid 444–456 sequence is shown in bold. Mutated amino acids are underlined. The NES mutants, GFP-HEG4A and GFP-HEGIL, were prepared as described in Materials and methods. (b) Quiescent MCF-7 cells were transfected with the indicated plasmids and then left unstimulated or stimulated with 10 nM estradiol for the indicated times. The percentage of cells with nuclear GFP protein was determined by fluorescence microscopy and shown graphically. For each experiment, at least 150 cells were scored. The results of different independent experiments were averaged; n represents the number of experiments. (c) Images of one experiment in panel b that shows the intracellular distribution of GFP-wtERα, GFP-ERα 4A, or GFP-ERα IL in MCF-7 cells treated for 1 h with estradiol. Bar, 5 μm. (d–f) Growing NIH3T3 fibroblasts were used. The Western blot in panel d shows that NIH3T3 fibroblasts are ERα negative. In the graph in panel d, cells were transfected with an ERE-Luc construct along with the indicated plasmids. After transfection, the cells were made quiescent and then left unstimulated or stimulated with 10 nM estradiol. Luciferase activity was assayed, normalized using β-gal as an internal control, and expressed as fold induction. (e) Cells were transfected with the indicated plasmids and made quiescent. The cells were left unstimulated or stimulated for 5 min with 10 nM estradiol. Lysates were analyzed for AKT activation using the anti–P-Ser-473 antibody (bottom). The nitrocellulose filters were then reprobed with anti-AKT antibody (top). (f) Cells on coverslips were transfected with the indicated plasmids and made quiescent. The cells were left unstimulated or stimulated for 18 h with 10 nM estradiol or 20% serum. In cells transfected with GFP-wtERα, the inhibitory action of Tat peptide on estradiol-induced BrdU incorporation was analyzed by including this compound (at 1 μM) to the cell medium (bars with asterisks). After in vivo pulse with BrdU, DNA synthesis was analyzed by immunofluorescence. In transfected cells, BrdU incorporation was calculated by the formula (percentage of BrdU-positive cells = [number of transfected-positive cells/number of transfected cells] × 100) and compared with BrdU incorporation of untransfected cells from the same coverslips. For each plasmid, data were derived from at least 500 scored cells. The results of several independent experiments were averaged; n represents the number of experiments. (b, d, and f) Means and SEM are shown.