Figure 2.

Heregulin reverses the anti-proliferative effect of tamoxifen in clonogenic and anchorage independent assays. (A) Clonogenic assays: MCF-7 cells cultured at low density in standard 60 mm tissue culture plates. Cells were treated with vehicle (control), estrogen [1 nM] (E2), tamoxifen [100 nM] (TAM), heregulin [10 ng/mL] (HRG) or tamoxifen and heregulin (HRG-TAM) and colonies enumerated after 12 days. *p < 0.001 comparing tamoxifen-treated and heregulin with tamoxifen treated cultures. (B) Anchorage-independent growth assays: MCF-7 cells in 0.35% bactoagar with the different treatments as above were grown on the bottom layer of 0.6% bactoagar in 35 mm tissue culture plates. Colonies were enumerated after 10 days. *P < 0.001 comparing tamoxifen-treated and heregulin with tamoxifen treatedcultures Graphs represent the mean colony number of nine dishes. Error bars indicate 95% CI. (C): Serum starved MCF-7 cells were treated with vehicle (control), tamoxifen [100 nM] (TAM), heregulin [10 ng/mL] (HRG) or tamoxifen and heregulin (HRG-TAM) for 24 hours and analyzed by flow cytometry. Each column represents the mean of the percentage of cells in G1 and S phase of the cell cycle from triplicate plates; error bars indicate 95%CI; * p < 0.001 comparing TAM and control; # p < 0.001 comparing TAM and HRG-TAM cells.