ERa Function by Image Analyses Figure 5. EGFR signaling and PRL array responses. PRL-HeLa cells transiently transfected with GFP-ER were pretreated or not with Tryphostin AG537 for 30 minutes before adding ethanol (vehicle), E2, EGF or E2 and EGF together for 2 hours. A. Promoter targeting: after fixing and counterstaining with DAPI, cells were imaged and the percentage of each cell population that showed visible accumulation at the array in response to treatment was calculated. Student t-test was performed for EGF treatment compared to each other treatments groups (* p,0.05, { p,0.03). B. Large-scale chromatin modification: the same images were acquired using high throughput microscopy and the array size was quantified as described in the Methods. Differences from respective vehicle treatment reaching statistical significance (p,0.05) are labeled by asterisks (*). C. Transcriptional activity: subsequent to ligand treatment, the cells were fixed and subjected to RNA FISH. The FISH signals at the array were quantified in .20 cells for each protein and treatment condition and graphed as the average total array-associated fluorescence 6SEM. Student t- test was performed for each treatment group compared to its control (* p,0.05), or to vehicle ({ p,0.05). doi:10.1371/journal.pone.0002286.g005 expressing GPF-ER-S118E showed no increase in the percentage of visible PRL-arrays, indicating that additional ER modification(s) may be important. We next analyzed E2- or EGF-induced large-scale chromatin modification and reporter mRNA transcription simultaneously in cells expressing GFP-ER phosphomutants. In cells transfected with GFP-ER-S118A, an increase in the level of mRNA was detected, which plateaued after 8 hours of E2 treatment, while the PRL- array was maximally decondensed after 4 hours (Figure 7A). In contrast, the EGF response was completely abolished in the presence of GFP-ER-S118A (Figure 7C). When cells were transfected with a GFP-ER-S118E phospho- mimic mutant, we observed a 1.5 fold increase in basal PRL-array size and a significantly higher basal transcript level (2.8 fold) in the absence of any stimuli (Figure 7B and D, time 0). Moreover, the responses of this mutant to E2 and EGF were very similar in terms of chromatin decondensation and transcriptional output (Figure 7 compare Panel B and D). The highly decondensed status of the chromatin before treatment was also sustained for over 24 hours. Somewhat similar to the wild type ER (see Figure 4C), levels of mRNA accumulation at PRL-arrays occupied by GFP-ER-S118E exhibit two pulses of FISH signal accumulation with major peaks at 30 minutes and 12 hours for estradiol, and 15 minutes and 8 hours for EGF (Figure 7B and D). The presence of the phosphomutant GFP-ERS118E induces a maximum peak of transcript levels for EGF at 15 minutes (p.0.05) (Figure 7B). The accumulation of mRNA at 8 hours after EGF exposure was similar to E2 at the same time point (p = 0.03) but was significantly lower compared to the second peaks induced by E2 with GFP-ER wt and GFP-ERS118E (p.0.05) (Figure 7). Discussion It has been well demonstrated that EGF signaling pathways modulates ER activity [3,14], although the molecular mecha- nism(s) is not clearly established. The technical complexity of cell- based assays for promoter binding, chromatin structure, and/or PLoS ONE | www.plosone.org 7 May 2008 | Volume 3 | Issue 5 | e2286