Estrogen Signaling in Breast Cancer Cells

Cells were grown in DMEM containing 10% charcoal-stripped FBS until 80% confluence, followed by treatments with phosphate buffered saline (PBS; control) or 5 or 10 nM β-estradiol (E2; Sigma-Aldrich; #E1024) in PBS for 4 h. For E2 treatment in the MCF7 cell line, both PBS and E2-PBS buffers contained 20 nM MLN4924 (a small molecule inhibitor of the Nedd8 activating enzyme) (Sigma-Aldrich; # 5054770001). After two washes with ice-cold PBS, the cells were used for RNA and protein isolation. The T47D cells were also cultured in DMEM containing 10% charcoal-stripped FBS and then treated with PBS, 100 nM bufalin (Sigma-Aldrich; #B0261) in PBS, or 5 μM gossypol (Sigma-Aldrich; #PHL83856) in PBS for 6 h, followed by additional treatment with or without 10 nM E2 for another 4 h. The treated cells were washed twice with ice-cold PBS buffer and used for RNA and protein isolation.