Supporting Information Figure S1 Assessment of ER ability to recruit coactivators to the promoter array. PRL-HeLa cells transiently expressing GFP-ER, were treated with 10 nM E2, 100 ng/ml EGF or ethanolic vehicle for 2 hours. Subsequent to ligand treatment the cells were fixed and endogenous coactivators (SRC-1, -3, BRG1, CDK9 and CyclinT1) detected by immunofluorescence (red). The presence of each coactivator at the promoter array is identified by accumu- lated signal above the level for the nucleoplasm, and representative images are illustrated here as a merge. The size bar is in microns. Found at: doi:10.1371/journal.pone.0002286.s001 (2.60 MB TIF) Figure S2 Stimuli withdrawal regulates large-scale chromatin modification: PRL-HeLa cells were transiently transfect with GFP- ER and then treated with 10 nM E2 or 100 ng/ml EGF for 30 min (time = 0). After this time the cells were washed 5 times with PBS and the medium replaced with medium without Phenol Red and 5% SDFBS. The cells were then fixed at the indicated times, stained with DAPI and imaged with the HTM in order to quantify the array size. Results are expressed as array size normalized to control cells obtained from three independent experiments. The graph representing the cells without replace- ment of the stimuli (full line) is indicated for comparison. The decay rates were as follows: E2 withdrawal = 0.29/h; EGF withdrawal = 0.16/h; EGF = 0.026/h. Found at: doi:10.1371/journal.pone.0002286.s002 (0.20 MB TIF) Figure S3 Assessment of ER ability to recruit RNA Polymerase II. PRL-HeLa cells transiently expressing GFP-ER, were treated with 10 nM E2, 100 ng/ml EGF, 10 nM 4HT or ethanolic vehicle for 2 hours. Subsequent to ligand treatment, the cells were fixed and endogenous RNA Polymerase IIo detected by References 1. Smith CL, O’Malley BW (2004) Coregulator function: a key to understanding tissue specificity of selective receptor modulators. Endocr Rev 25: 45–71. 2. 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Found at: doi:10.1371/journal.pone.0002286.s003 (4.17 MB TIF) Acknowledgments The authors wish to thank JH Price (Vala Sciences, San Diego) for invaluable long-term support in bringing this project to fruition, A Szafran for HTM analysis, L Pasini for help with imaging, and ED Jones for statistical advice. The authors gratefully acknowledge C Haueter, KM Antalis, and D Townley for excellent technical support. All imaging was performed in the Department of Molecular and Cellular Biology Integrated Microscopy Core (Center for Reproductive Biology, O’Malley; Dan L. Duncan BCM Cancer Center, Osborne; John S. Dunn Gulf Coast Consortium for Chemical Genomics, Davies and Mancini). Current addresses: V.B., European Molecular Biology Laboratory, Mouse Biology Unit, via Ramarini, 00016 Monterotondo, Italy, ; valeria.berno@embl.it; L.A., Inserm, U693, 63 rue Gabriel Pe´ri, F-94276, Le Kremlin-Biceˆtre, France, ; larbi.amazit@u-psud.fr. 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(2005) Growth factors change nuclear distribution of estrogen receptor-alpha via mitogen-activated protein kinase or phosphatidylinositol 3-kinase cascade in a human breast cancer cell line. Endocrinology 146: 4082–4089. PLoS ONE | www.plosone.org 12 May 2008 | Volume 3 | Issue 5 | e2286