Figure 1

Hormone-independent effects of ER on the proliferation of hormone-sensitive MCF-7 cells. In all the experiments, MCF-7 cells were first cultivated in phenol red free DMEM containing 5% charcoal-stripped FBS for 48 hours to deplete hormone. EGR3 mRNA levels in the cells were measured by real time RT-PCR after a brief (8 h) treatment with vehicle (No ligand), E₂ (1 nM) or OH-Tam (100 nM) (A). Cells were also treated with vehicle (No ligand) or E₂ (10 nM) for 30 minutes and ligand-dependent activation of ER was analyzed by western blot using a specific antibody to detect phosphorylation at ser¹¹⁸ of ER; GAPDH was probed as a loading control (B). Hormone-depleted MCF-7 cells were transfected with either ER siRNA or control siRNA and maintained in hormone-depleted conditioned media without further replenishment of the media. Twenty-four hours after transfection, cells were treated with vehicle (No ligand), E₂ (1 nM) or OH-Tam (100 nM); the treatments were repeated every 48 hours without changing the media. Following the treatment, viable cells were counted daily for six days by the Trypan Blue dye exclusion assay (C). In parallel, cells were harvested on each day of the treatments and total RNA extracted from them; the mRNA for ER was measured by real time RT-PCR and the values were normalized to those for GAPDH (D). In addition, cells were harvested in parallel on each day of the treatments for western blot analysis using antibody to ER; GAPDH was probed in each blot as a loading control (E). P-values for the differences noted in the text were ≤0.001.