Experimental design and microarray data selection of the time-course analysis of estrogen response in T-47D cells. (a) Cells were starved of serum and estrogen for 24 h before treatment with dimethysulfoxide (DMSO; carrier control), 17β-estradiol (E2), and E2 in combination with ICI 182,780 (ICI) and cycloheximide (CHX). Samples were taken at the 16 time points indicated. (b) Procedure for identifying direct ER targets. Data selection for estrogen-responsive genes was based on p-value cutoffs (p ≤ 0.052) and magnitude of response (at least 1.2-fold change in the same direction for three time points). ICI sensitivity and CHX insensitivity were also determined by statistical measures, p ≤ 0.058 and p ≥ 0.24 respectively. Cutoff values were informed by expression profiles of cathespsin D and progesterone receptor, both known to be regulated by ER.