Figure 3

Effect of fulvestrant on cell cycle phase distribution in hormone-depleted MCF-7 cells. Hormone-depleted MCF-7 cells were treated with vehicle control or fulvestrant (100 nM) and maintained in hormone-depleted conditioned media without further replenishment of the media. The treatments were repeated every 48 hours for up to five days. (A) Cells were harvested after three and four days of treatment as described above and total RNA extracted from them. The mRNA for ER was measured by real time RT-PCR and the values were normalized to those for GAPDH. (B) Cells were harvested after three and four days of treatment as described above for western blot analysis using antibody to ER. GAPDH was probed as a loading control. (C) The cells treated as above were harvested on days 3 and 4 for flow cytometry analysis to determine their cell cycle phase distribution. (D) On Day 4 of the above treatment, cells were harvested to measure the proportion of apoptotic cells by Annexin V staining. P-values for the differences noted in the text were ≤0.001.