ERa Function by Image Analyses r Figure 3. Large-scale chromatin decondensation of the PRL-array. A. PRL-HeLa cells expressing GFP-ER were treated for different times with ethanol (vehicle), E2 or EGF. After fixing and counterstaining with DAPI, cells were imaged and the array size was quantified using high throughput microscopy. B. This panel represents earlier time points to show the slower effect of EGF in inducing maximal decondensation of the array. Data represent the mean 6SEM of three different experiments. B. PRL-HeLa cells transiently expressing GFP-ER were imaged live at 37uC under constant perfusion of fresh medium (plus 100 ng/ml EGF after time 0). Image stacks were recorded every four minutes and are presented as projections. The value indicates the area of the array (mm2) shown in the image. Addition of EGF caused chromatin decondensation at the array within 30 minutes. doi:10.1371/journal.pone.0002286.g003 (Figure 5B). Importantly, however, there was an additive effect on transcript accumulation at the array (p.0.05, Figure 5C), which agrees with several previous studies of reporter gene activation [3,12,23]. However, when PRL-HeLa cells were treated with AG537 prior to addition of E2 and EGF, transcript levels were comparable to those obtained with E2 treatment only (p.0.05, Figure 5C). Since the MAPK cascade is implicated in ER transcription regulation [15,16], we treated PRL-HeLa cells with E2 or EGF in the presence or absence of two different inhibitors of the MAPK pathway; PD98059 and UO126. Among all cells expressing a low level of GFP-ER, both inhibitors markedly decreased the percentage of cells with labeled arrays (66% reduction; Figure 6A). Conversely, inhibiting the MAPK pathway did not alter E2-dependent recruitment of ER to the PRL promoter array (data not shown), clearly showing that E2 and EGF operate through distinct molecular mechanisms to activate the transcrip- tional activity of ER. Serine 118 in ER-mediated PRL array responses Serine 118 in the ER is a target residue of EGFR initiated signal transduction cascades including the MAPK pathway [14]. Conversion of this residue to a non-phosphorylable alanine (GFP-ER-S118A) strongly reduced the number of cells with ER- positive PRL-arrays after EGF treatment (75% reduction, Figure 6B). However, this mutation had only a minor effect on E2-dependent PRL-array targeting (20% reduction) (Figure 6B). Conversely, replacement by glutamic acid (S118E), which mimics phosphorylation, targeted the PRL-array similarly to wild-type ER under both conditions (E2 or EGF). Strikingly, control cells Figure 4. ER transcriptional activity at the promoter array. PRL-HeLa cells transiently expressing GFP-ER were treated with E2, EGF, 4- hydroxytamoxifen (4HT) or ethanolic vehicle for the indicated time. Subsequent to ligand treatment the cells were fixed and subjected to an RNA FISH protocol using a biotinylated dsRED2 probe followed by fluorescent-tagged streptavidin. A. Representative images of a single cell for each treatment. The presence of transcripts at the promoter array is identified by accumulated signal above the level for the nucleoplasm. The inset values (red type) represent the amount of transcript at 2 hours, relative to vehicle controls. B. Representative image of PRL-HeLa cells transfected with GFP- ER (green) exemplify the heterogeneity of ER expression levels. The cells show the RNA FISH signal associated with the array in the cell population (red signal) (I). The nuclear GFP-ER mean of fluorescence and RNA FISH array signal were plotted for vehicle- and E2-treated cells. Each symbol represents the measurements from a singe cell (II). C. To quantify FISH signal over 24 hours the total intensity of signal at the array (minus background signal) was determined by cumulative summation of 20 planes. Data represent the mean 6SEM of three different experiments graphed as fold induction over mean time-matched vehicle-treated control cells. D. This panel represents the earlier time points for E2 and EGF treatment. Fold activation between EGF and E2 at 30 minutes was significantly different, with a p value of 0.03. doi:10.1371/journal.pone.0002286.g004 PLoS ONE | www.plosone.org 6 May 2008 | Volume 3 | Issue 5 | e2286