FIGURE 6.
Effects of ERα and ERαEBD on proliferation and cycle distribution of U-2OS cells. A, synthesis of ERs. Cells were infected for 48 h with the parent recombinant adenovirus (Ad5) at m.o.i. 50, a recombinant adenovirus bearing cDNA for ERα at 40 m.o.i. or ERαEBD at 50 m.o.i. The total m.o.i. was adjusted to 50 by supplementing with parent Ad5. Cell extracts (10 μg) of infected cells were subjected to WB using the horseradish peroxidase-conjugated monoclonal FLAG antibody. Molecular mass in kDa is indicated. B, cell extracts (10 μg) were also subjected EMSA in the absence (-) or presence (+) of a FLAG antibody (Ab). ERE indicates the unbound and ER-ERE depicts the ER-bound radiolabeled ERE. C, assessing the ability of an ERα to binding to E2 in situ. Cells infected with recombinant adenoviruses for 48 h in the absence of a ligand were incubated in medium containing 10-7 m of [3H]E2 for 1 h. Medium containing the radioactive [3H]E2 was removed. Cells were then extensively washed with fresh medium and dislodged. Radioactivity retained in cells was quantified by scintillation counting. The specific retention of [3H]E2 was assessed by the co-incubation of cells with 10-6 m ICI. The graph represents the means ± S.E. of three independent experiments performed in duplicate. D, infected cells with recombinant adenoviruses in the absence (-) or presence (E2) of 10-9 m E2 for 48 h were subjected to FACS. Results, presented as the means ± S.E. of three independent experiments, are the depiction of a cell population in G1, G2, and S phases. E, cells were infected with recombinant adenoviruses bearing none (Ad5) or an ERα cDNA. Cells were maintained in the absence (-) or presence of 10-9 m E2 (E2) for 6 days. At the termination, cells were counted. The means ± S.E., which are three independent experiments performed in duplicate, indicate relative change in cell number compared with those observed in cells infected with the parent Ad5 in the absence of E2, which is set to 1. Asterisk indicates significant change.