E2 induces complete relocalization of ERα in established T47D:A18/PKCα colonies after 10 days. A. T47D:A18/neo colonies (neo) and T47D:A18/PKCα colonies (PKCα) colonies were immunostained for ERα (green) and nuclei (blue). All images were acquired and processed using parameters described in materials and methods. Colonies were grown for 10 days then treated for 10 days with vehicle (EtOH, 0.1%), E2 (10-9 M), 4-OHT (10-7 M) or RAL (10-7M). Scale bar = 20 μm. B. Expression of ERα in whole cell colony lysates. Molecular weights of ERα and β-actin are 67 kDa and 42 kDa, respectively. Values represent β-actin-normalized ERα expression relative to T47D:A18/neo E2-treated colonies.