ERa Function by Image Analyses Figure 2. Role of AF-1 and AF-2 ER domains in promoter binding. Representative images of PRL-HeLa cells transiently expressing the indicated deletion mutants and treated with either ethanolic vehicle, E2 or EGF for 2 hours (see Table 1). The inset images are representative of fluorescence images observed in a minority of cells. The size bar is in microns. All images were captured so as to optimize the intensity of the GFP-ER. doi:10.1371/journal.pone.0002286.g002 treated with antagonists (4HT or ICI) demonstrated undetect- able association of these co-regulators. Based on our set of immunological reagents, these data indicate that EGF signaling through ER recruits a similar set of coregulators at the PRL array. Temporal Large-Scale Chromatin Modification To further explore E2 and EGF stimulation of ER, we performed time-course treatments (0–24 hours) and cytomorpho- metric analysis of large-scale chromatin modification by high- throughput microscopy (HTM). This new quantitative imaging Table 1. Promoter targeting and chromatin status in PRL-HeLa for ER deletion mutants. vehicle E2 EGF wt 60% (62.8) decondensed 100% decondensed 78% (64) decondensed *p,0.03 versus respective wt. {p,0.03 versus wt-EGF. doi:10.1371/journal.pone.0002286.t001 251–595 1–282 179–595 0% 0% 0% 0% 0% 0% 22.5% (63.5)* decondensed 89.5% (60.7)*,{ decondensed 19% (62.8)* decondensed 1–534 0% 100% condensed 3% (60.01) condensed PLoS ONE | www.plosone.org 3 May 2008 | Volume 3 | Issue 5 | e2286