approach [21] is a powerful tool that allows us to examine only those cells expressing a physiological level of GFP-ER (See Materials and Methods). In concert with previous live cell results [19], E2 treatment is associated with a rapid decondensation of the PRL-array that reaches its maximum expansion in ,15 minutes and, as shown in (Figure 3A), plateaus for at least 24 hours. In contrast, treatment of GFP-ER expressing cells with EGF resulted in a transient decondensation of the array, with full recondensation in about 2 hours (Figure 3A). A more detailed analysis of the early time period during expansion and contraction of the array is shown in Figure 3B. Note that EGF induced large-scale chromatin modification was slower than E2; ,30 minutes for peak expansion with EGF compared to ,15 minutes for E2. This EGF result is illustrated by time-lapse imaging of live cells shown in Figure 3C. We next determined if these stimuli-induced changes in large- scale chromatin persist upon removal of E2 or EGF. Results showed that the area of the PRL-array gradually decreased, and reached basal (no treatment) condensation by approximately 2 hours after removal of E2 (Figure S2). Upon removal of EGF, the array experiences a steeper rate of condensation compared to E2 (Figure S2). Note that both E2 and EGF failed to change the size of the array in non-transfected cells, in which the array was immunolabeled with antibodies specific for RNA polymerase II or dimethlylated histone H3 (K4) (data not shown). ER-mediated transcriptional activity at the single cell level To examine the relationship between array size and transcrip- tional responses to EGF, E2 or 4HT, we next used RNA FISH to measure dsRED2skl mRNA accumulation at the array (Figure 4A). Restorative deconvolved image stacks from GFP- ER and hybridization signals (array-associated transcripts) were collected and quantified as a measure of co-localizing reporter mRNA. First we examined the entire range of the expression within the transiently transfected cell population to determine if there was a cell-to-cell correlation between GFP-ER expression levels and the quantified FISH signal. In the PRL-HeLa cells that were non-transfected (fluorescence mean intensity was ,,500, Figure 4B, yellow circles), there was a low level of FISH signal detectable at the array (Figure 4A, inset value). Cell gating based upon ER expression level indicated a suppression of reporter transcript accumulation in cells overexpressing the receptor (GFP-ER mean intensity in the nucleus ,.8000, Figure 4B, blue circles; intensity value .1.5fold compared to MCF-7 cells). In the remaining cells with low levels of GFP-ER (,400062000, Figure 4B, red circles), FISH signals were not linearly dependent on fluorescence (receptor) levels, suggesting possible involvement of both expression level and an asynchronous cell population [22]. These observations led us to analyze cells with low levels of fluorescence (mean intensity,,6000) in the following experiments. In the E2 and EGF-treated cells, the mRNA transcripts at the PRL-array dramatically increased in comparison with both of the non-transfected cells and the vehicle treated cells (Figure 4A see the corresponding inset values). Treatment with 4HT repressed the accumulation of reporter transcripts at the PRL-array (see the arrows in the GFP-ER panel and the corresponding RNA FISH panel, Figure 4A) to levels below that of non-transfected cells or vehicle controls. In all cases, the transcriptional activity at the PRL-array correlated with the detection (or absence for 4HT- treated cells) of endogenous RNA polymerase II, which co- localized with the array (Figure S3). ERa Function by Image Analyses We then ask: do the timing differences in the PRL-array condensation/decondensation upon E2 or EGF treatment (See Figure 3A) correlate with changes in transcript accumulation at the array and, more precisely, is peak chromatin decondensation at the PRL-array co-incident with maximum co-localizing transcripts? To address this question, we performed quantitative RNA FISH at regular time intervals (0–24 hours) after treatment of PRL-HeLa cells expressing GFP-ER with E2 or EGF. Results showed that EGF treatment leads to a strong, single pulse of transcript accumulation at the array ((2.8 fold of vehicle) Figure 4C), consistent with its ability to induce an increase in the area of the PRL-array (Figure 3A), both of which demonstrate similar temporal dynamics. Maximal FISH signal peaked at 30 minutes of treatment, declined sharply (approxi- mately 40%) at one hour post treatment, and from there they gradually returned to basal (vehicle levels) by 4 hours (p.0.05, Figure 4C). By measuring the decay rate (l) of the graph from an exponential curve fit, we observed that the change in array size was slower when compared with the reduction of transcriptional activity (l = 0.026/h and l = 0.063/h respectively). In E2-treated PRL-HeLa cells expressing GFP-ER, a temporal analysis of RNA FISH showed a dramatically different pattern of reporter mRNA accumulation at the array (Figure 4C). While the PRL-array remained maximally decondensed (Figure 3A), the levels of reporter gene mRNA showed cyclical fluctuations over a 24-hour period (Figure 4C). Similar to the EGF treatment, FISH signals at the PRL-array peaked at 30 minutes post E2 treatment reaching 3.5 fold over basal levels, which is significantly higher (p = 0.03) than that induced by EGF (2.8 fold). From this point on in the 24-hour treatment period, the E2 transcription response at the PRL array was very different. The decline in FISH signal showed a slower rate of decrease compared to EGF reaching basal levels in ,2–3 hours. At arrays that are fully decondensed (Figure 3A), E2 treatment induced a second peak of mRNA accumulation around 4 hours post-treatment (2.5 fold higher than non-treated control cells). By 2–3 hours after this point, transcript levels again decreased to vehicle-treated values. Interestingly, 24 hours after beginning E2 treatment, mRNA levels at the array again increased to a level ,50% higher than control cells. Antagonist treatment (4HT) rapidly (within 15 minutes) repressed transcriptional activity, with no fluctuations (Figure 4C) con- comitant with tight array condensation [19]. Collectively, these results identify an agonist-ligand-specific wave-pattern of mRNA accumulation at the PRL array by sustained E2 treatment versus a single pulse pattern of mRNA accumulation during EGF activation. Cell Signaling and ER-mediated transcription To further test EGF-dependent pathways in ER-mediated activation of the PRL array, we used an inhibitor of EGFR autophosphorylation (Tyrphostin AG537) in our assays (Figure 5A–C) described above. Among all cells expressing GFP-ER, EGF plus AG 537 treatments resulted in a 66% reduction (relative to EGF alone control) in array targeting (Figure 5A). Furthermore, array size and mRNA accumulation among the few (20%), positive cells) was similar to the control vehicle-treated cells (Figure 5B and C), indicating that both large-scale chromatin decondensation and reporter gene induction by ER were inhibited. As expected, the inhibitor had no effect on the GFP-ER promoter targeting by E2 (Figure 5A), or chromatin decondensation (Figure 5B) and increased transcript accumula- tion (Figure 5C). EGF used in combination with E2 induced an increase in array size similar to that obtained with each factor separately PLoS ONE | www.plosone.org 4 May 2008 | Volume 3 | Issue 5 | e2286