Figure 3

ER-mediated transactivation and ligand sensitivity is altered in TAMR ^M cells. (A) MCF7 and tamoxifen-resistant cells derived from MCF7 (TAMR^M) cells were transfected with ESR1-directed small interfering RNA (siRNA) or scramble and total cell count was monitored after 5 days. Western blot of ER knockdown is presented. (B) MCF7 and TAMR^M cells were transfected with an ERE-tk-luc plasmid and treated with either vehicle (DMSO); 0.01 or 0.1 nM estradiol (E2); 10 or 100 nM 4-hydroxy tamoxifen (Tam); 10 or 100 nM fulvestrant (Ful) for 20 hours and assayed for luciferase activity (C) MCF7 and TAMR^M cells were transfected with ESR1-directed siRNA and assayed for mRNA expression of estrogen receptor (ER) target genes. mRNA expression is normalized to scramble control for each cell line. (D) MCF7 and TAMR^M cells were incubated in phenol red-free media supplemented with 5% charcoal dextran stripped serum (CDSS) for 72 hours after which 0 nM or 0.1 nM E2 was added for 24 hours and mRNA expression of depicted genes was quantified and represented as fold change upon E2 stimulation versus no E2. For graphs, averages are presented with error bars indicating the standard error of the mean.