MCF7 cells have acquired resistance to tamoxifen. (a) The effects of tamoxifen on the cell cycle progression. MCF7-WT and MCF7-TamR cells were seeded in the presence of 5 μM 4-hydroxytamoxifen (shown as 5 μM Tam) or ethanol vehicle, and total DNA content was determined by 7-AAD staining after 72 h. Results are shown as the average and s.d. of three biological and two technical replicates each. *P<0.05, **P<0.001, ***P<0.001, determined by a two-sided t-test, hereafter. (b) The effects of tamoxifen on cell viability. MCF7-WT and MCF7-TamR were untreated or treated with tamoxifen, and viable cells were counted by a luciferase-based viability assay after 72 h. (c) Proliferation rate of WT and TamR MCF-7 cells. Proliferation rates were measured after 48 and 72 h by direct counting (left) and with a luciferase-based viability assay (right). (d) ERE activity was measured after stimulating cells with E2. The results are shown as average of three biological and four technical replicates each. (e) Western blot validation of molecular markers of tamoxifen resistance, with β-actin as the loading control.