Evaluation of ERα activity and expression. (A) MCF-7 human breast cancer cells were transfected with the ERE-TK-LUC plasmid containing the luciferase reporter gene under the estrogen responsive element (ERE) and co-transfected with β-galactosidase plasmid. The media was removed 24 h after transfection and 10 nM of 17β-estradiol was used to induce ERα activity. Synthesized compounds at 10 μM concentration or vehicle control (cont) was added to phenol-free DMEM supplemented with 10% steroid-free serum (Hyclone). The luciferase and β-galactosidase activities were determined in 24 h. The luciferase activity was calculated in relative units evaluated as the ratio of the luciferase activity to the galactosidase activity. *P < 0.05 vs. E2-treated cells. (B) MCF-7 cells were treated with or without tamoxifen or compound 4a. The cell samples were subjected to Western blot analysis of ERα and α-tubulin as the loading control; Densitometry for ERα/α-tubulin ratio was carried out using ImageJ software with the densitometry protocol provided by The University of Queensland. *P < 0.05 vs. control cells, #P < 0.05 vs. tamoxifen-treated and control cells.