Figure 5.

FKHR nuclear export: regulation by estradiol and a role in hormone-induced DNA synthesis in MCF-7 cells. Quiescent MCF-7 cells on coverslips were used. (a) Cells were transfected with the indicated plasmids then left unstimulated or stimulated for 24 h with 10 nM estradiol. After in vivo pulse with BrdU, DNA synthesis was analyzed by immunofluorescence and BrdU incorporation was calculated as in Fig. 4. (b) Cells were transfected with the indicated plasmids then left unstimulated or stimulated with 10 nM estradiol for the indicated times. Endogenous ERα localization as well as expression of GFP, GFP-FKHR wt, or GFP-FKHR AAA mutant was monitored by confocal microscopy. Cells that fell into the category of exclusively ERα nuclear fluorescence were scored, and data were expressed as a percentage of transfected cells. (c) Cells were cotransfected with the indicated plasmids then left unstimulated or stimulated with 10 nM estradiol for 60 min. Localization of GFP-FKHR wt, Myc-HEG0, or Myc-HEGIL mutant was monitored by confocal microscopy. Cells that fell into the category of exclusively FKHR nuclear fluorescence were scored and the data were expressed as a percentage of cotransfected cells. For each experiment in panels a–c, data were derived from at least 500 scored cells. The results of several independent experiments were averaged; n represents the number of experiments. (d) Images from one experiment in b or c are shown. They represent the staining of endogenous ERα (red) in MCF-7 cells expressing GFP-FKHR wt (left, green) or the mutant, GFP-FKHR AAA (middle, green), and treated for 60 min with estradiol. (right) The staining of Myc-tagged NES-ERα mutant (red) in MCF-7 cells coexpressing GFP-FKHR wt (green) and treated for 60 min with estradiol. Merged images are also shown on the bottom. Bar, 5 μm. (e) The cells were cotransfected with ERα siRNA (ERα siRNA) or nontargeting siRNA (nt siRNA) and GFP-FKHR wt. The cells were then left unstimulated or stimulated with 10 nM estradiol for the indicated times. GFP-FKHR wt localization was monitored by confocal microscopy. Cells that fell into the category of exclusively GFP-FKHR wt nuclear fluorescence were scored and data were expressed as a percentage of transfected cells. Data were derived from at least 200 scored cells. The results of two independent experiments were averaged. The blot in panel e confirms the silencing of ERα in MCF-7 cells transfected with ERα siRNA (top). The bottom shows the blot of loading proteins revealed using the anti-tubulin antibody. (a, b, c, and e) Means and SEM are shown.