approach described here increases in terms of automation and speed, the resultant avalanche of terabytes of image data per experiment will provide unprecedented data-mining opportunities, and challenges. Current studies focusing upon a spatiotemporal fingerprint of receptor and coregulator levels at the PRL locus, or other regulated gene arrays, should yield additional key insights into the orchestration of the molecular and cellular events involved in gene expression. Materials and Methods Cell culture For all experiments using PRL-HeLa, cells were grown a minimum of two days in hormone-free medium containing stripped and dialyzed fetal bovine serum. Transient transfection of ER and ER mutant expression vectors was performed using the Bio-Rad Transfectin reagent following the manufacturer’s proto- col. After transfection the medium was replaced with ligand containing media and treated for the desired length of time. Immunolabeling Antibody labeling was performed as described previously [19,49] using 4% formaldehyde fixation (30 min) and indirect labeling with Alexa 488, 546 or 633(Molecular Probes) conjugated secondary antibodies. DAPI (1 mg/ml) was used to label DNA prior to mounting in Slow Fade (Molecular Probes). Affinity- purified rabbit antibodies to SRC-1 and SRC-3 were a kind gift from Dr. Jeimin Wong; CDK9, CyclinT1, BRG-1 and RNA Pol II large subunit were obtained from Abcam or Upstate Biotechnol- ogy and all used at 1 mg/ml for 1 hr at room temperature or overnight at 4uC. RNA FISH The methods used here, including procedures for non-isotopic probe preparation and fluorescent in situ hybridization, have been published in detail [50] Briefly, coverslips with adherent cells were rinsed twice in PBS, dipped in cytoskeleton (CSK) buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES, pH 6.8) [51], extracted on ice for 5 minutes in CSK buffer containing 0.5% Triton X-100 and 2 mM vanadyl-ribonucleoside complex (VRC; Gibco-BRL), rinsed in CSK/VRC, fixed in 4% parafor- maldehyde/PBS for 10 minutes, rinsed again in PBS and stored in 0.4% paraformaldehyde at 4uC until use. Probes substituted with biotin-labeled deoxynucleotides were made by modifications to standard nick translation procedures [50] Hybridization to RNA was carried out at 37 C in standard buffers containing 5 mg/ml probe and 50% formamide overnight. After incubation, samples were rinsed in a series of SSC buffers, assayed for biotin using streptavidin, Alexa Fluor 546 conjugate (Molecular Probes, Eugene, OR) and rinsed in a series of PBS washes. Cells were counter-stained with 1 mg/ml DAPI (Sigma, St. Louis, MO). Coverslips were then mounted on slides in Slow Fade (Molecular Probes). To quantify FISH signals, a 20 plane 1286128 (pixels), 0. 2 mm Z-stack was collected (constant exposure). Images were acquired on a Deconvolution Microscope, deconvolved and sum projected. The RNA FISH signals were quantified by using MetaMorph software (Universal Imaging, Downingtown, Pa.) after subtraction of the background nuclear fluorescence as previously described [40].Then, the integrated total RNA FISH intensity was calculated for each condition and normalized to the level of integrated total RNA FISH intensity in untreated cells to obtain the relative RNA FISH intensity. Linescans were created using SigmaPlot (Systat Software, Inc., San Jose, CA). ERa Function by Image Analyses Fixed Cell and Time-Lapse Imaging The XFP-fusions and immunofluorescently labeled cells were imaged using a DeltaVision Restoration Microscopy system (Applied Precision, Issaqua, WA) and applying a constrained iterative deconvolution process. Whole nuclear volumes were collected at 0.2 mm Z steps and images from select focal planes or 3D projections were imported into Adobe PhotoShop. The Histogram adjustments were made relevant to negative controls, which routinely included non-transfected cells and/or omission of primary antibodies. Live imaging was performed by collecting short Z-stacks (,5–10 focal planes at 300 nm increments); neutral density for the green channel was set at 50% and the images were binned 262. Typical exposures were for ,1 sec, and time points from 3–10 minutes per stack. Projected images from each time point were used to create a QuickTime movie. Cells for live imaging were grown in 35 mm Delta T dishes (Bioptechs, Butler, PA) and secured to a stage adapter for temperature control. HEPES-buffered media was gassed overnight in a 5% CO2 incubator, and circulated through the Delta T dish using a Bioptechs peristaltic pump and inflow/outflow tubing. The temperature was controlled to 37uC (6,0.1 degree); a Bioptechs objective heating collar was also used (also at 37uC). The Delta T dish was covered with a black plastic lid, with room for input/ output tubing. For time-lapse imaging, a 636 objective (NA = 1.4) was used. Quantitative Image Analyses by HTM The PRL-HeLa cells transiently expressing GFP-ER were treated with E2, 4HT, or EGF for the time indicated, fixed, and DAPI stained. The cells were imaged using the Cell Lab IC 100 Image Cytometer (Beckman Coulter) with a Nikon 406 Plan S fluor 0.90 objective. Two channels were imaged: channel 0 (DAPI) was used to find the focus and nuclei and channel 1 was used to image GFP-ER. A proprietary algorithm (GPCR) developed at Beckman Coulter was used to identify and quantify the GFP-ER targeted PRL-array. The parameters for the GPCR algorithm were: object scale = 30 and minimum peak height = 10. Foci identified by the GPCR algorithm are masked. The area of the mask in pixels is the measure of PRL-array size. Channel 1 was offset 2 mm from the DAPI focus for cells in all treatment conditions. This offset provided the greatest number of in focus arrays identified by the GPCR algorithm. After image acquisition and application of the GPCR algorithm the total cell populations for each treatment were progressively filtered (gated) using the same criteria. Nuclei clusters, mitotic cells, and apoptotic cells were filtered from the total cell population using an intersection of DNA content and DNA clusters gates. In addition, low GFP-ER expression and low aggregate number gates were generated and applied to produce the final cell population to be analyzed. The maximum fluorescence detected of endogenous ER in MCF-7 cell line is determined as total fluorescence in the cell. The maximum threshold for exogenous ER expression detected by immunofluorescence in PRL-HeLa cells is conservatively set to 2 times the endogenous level. The corresponding range for green fluorescence of exogenous GFP- ER is determined and used for subsequent imaging. From the final population of cells, the array size was determined using the GPCR mask. The images and masks were visually inspected for accuracy. The data was imported into SigmaPlot via an Excel Spreadsheet and fit to a single exponential decay to obtain the dissociation rate constant. Unpaired Students t-tests assuming equal variance were performed to determine statistical signifi- cance (two-tailed, p,0.05). PLoS ONE | www.plosone.org 11 May 2008 | Volume 3 | Issue 5 | e2286