Comparison of HER2, EGFR and ERα expression between SKBr3 and other breast cancer cells. MCF-7 cells were grown in estrogen-free RPMI medium for four days then treated with either EtOH control or 1 nM E₂ for two more days before harvest. Other cells were grown in medium as described in materials and methods. Fifty micrograms of total proteins were used for western blot analysis for HER2, EGFR and ERα. The β-actin was also examined as a loading control.

The effects of ERα expression and estrogen/antiestrogen treatment on the proliferation of SKBr3 cells. SKBr3 cells were infected by Adeno-X Tet-off and Ad-TRE-ERα in the presence (+Dox) or absence (−Dox) of 1 μg/ml doxycycline, treated by the 0.1% EtOH (v/v), 1 μM fulvestrant (ICI), 1 μM 4-hydroxytamoxifen (4OHT) or E₂ (at final concentration of 1 nM or as indicated in the graph) and harvested for DNA quantification. (A) Growth with different ER ligands treated for six days. (B) Dose-dependent growth with various E₂ concentrations treated for six days. (C) Time-dependent growth with cells harvested every 2 days after treatment. The samples with a statistically significant difference (p<0.05 by t-test) from the +Dox/EtOH control were marked with a “*”.

Modification of p21^Cip1/Waf1, pRb and E2F1 by ERα/E₂ in SKBr3 cells. SKBr3 cells were infected, treated and harvested as in Figure 4. Protein was extracted for western blot analysis (A) and RNA was prepared for real-time RT-PCR analysis to detect E2F1 (B), p21^Cip1/Waf1 (C) or pRb (D) as described in Figure 2. The samples with a statistically significant difference (p<0.05 by t-test) from the +Dox/EtOH control were marked with a “*”.

The Tet-off adenoviral system to express ERα in SKBr3 cells. (A) SKBr3 cells were infected with Ad-CMV-GFP and observed 24 hours after infection with a TE300 fluorescence microscope (Nikon Instruments, Melville, NY). (B) SKBr3 cells were co-infected by Adeno-X Tet-off and Ad-TRE-ERα in the presence of doxycycline at various concentrations. The cells were harvested 48 hours after infection and total protein was extracted for western blot. (C) SKBr3 cells infected by Adeno-X Tet-off and Ad-TRE-ERα in the presence (+Dox) or absence (−Dox) of 1 μg/ml doxycycline were transfected with 5xERE-firefly-luciferase and TA-Renilla-luciferase plasmids. The cells were harvested for dual luciferase activity assay after 48-hour treatment with the compounds as indicated. The ratio of firefly luciferase vs Renilla luciferase activities were plotted and the number of the +Dox/EtOH sample was arbitrarily set to be 1 for easy comparison. (D) SKBr3 cells infected by Adeno-X Tet-off and Ad-TRE-ERα in the presence (+Dox) or absence (−Dox) of 1 μg/ml doxycycline were treated with 0.1% EtOH, 1 μM fulvestrant (ICI) or 1 nM 17β-estradiol (E2) for 48 hours. The total RNA was extracted for real-time RT-PCR analysis of PS2 or PR (E) against endogenous control 36B4 using a relative standard curve generated by 10-fold serial dilution of MCF-7 cDNA. The value of the +Dox/EtOH sample was arbitrarily set to be 1 for easy comparison.

Cell cycle analysis of SKBr3 cells expressing ERα. SKBr3 cells were infected by Adeno-X Tet-off and Ad-TRE-ERα in the presence (+Dox) or absence (−Dox) of 1 μg/ml doxycycline, treated by 0.1% EtOH, 1 μM fulvestrant (ICI) or 1 nM E₂ for 2 days and harvested for cell cycle analysis. (A) Flow cytometry analysis of cell cycle distribution. (B) Percentage of cells at G0/G1 cell cycle from three independent experiments. The samples with a statistically significant difference (p<0.05 by t-test) from the +Dox/EtOH control were marked with a “*”.

The effects of ERα expression and estrogen treatment on the expression of HER2 and EGFR in SKBr3 cells. SKBr3 cells were infected, treated for 2 days or 6 days then harvested for protein extraction and western blot analysis.