role in the expression of these genes which are related to cell proliferation and puerarin could suppress these key genes expression by inhibiting ERK1/2 activation. In conclusion, the total binding ability of puerarin to ERs on viable ESCs is around 1/3 that of E2, puerarin suppresses proliferation of ESCs induced by E2-BSA, partly via impeding a rapid, non-genomic, membrane-initiated ERK pathway, and down-regulation of cyclin D1, cox-2 and cyp19 are involved in this process. Puerarin may represent a potentially new approach in the medical treatment of endometriosis. Materials and Methods Sample Collection and Cell Isolation Tissues were obtained from patients, with ovarian endome- triotic cysts, undergoing laparoscopy. For all patients included in this study, pathology analysis was carried out on one part of the biopsy to confirm the presence of endometriotic tissue. The mean age of the subjects was 37.665.8 years (range 27–49); all subjects had regular menstrual cycles and had been independent of hormonal treatment for at least 3 months before surgery. The study was approved by the Committee on Human Research of Changhai Hospital, and written informed consent was obtained from all subjects. Specimens were collected, and ESCs were purified, cultured and identified as previously described [36]. Competitive Estrogen Receptor Whole ESCs Binding Assay ESCs were seeded at a density of 26104 cells/well in 96-well plates with phenol red-free DMEM (Dulbecco’s modified Eagle’s medium; PAA, Linz, Austria) supplemented with 10% charcoal- stripped fetal bovine serum (CSFBS) (Biological Industries, Israel). After cultivation at 37uC in a 5% CO2 atmosphere for 4 d, competitive binding assays were performed on whole cells using a previously described method [37]. The dissociation constant (Kd) of the ER and [2,4,6,723H]-Estradiol ([3H]-E2; PerkinElmer, Massachusetts, USA) complex was measured with nonlinear regression techniques. We then used the Kd value of labeled E2 to determine the IC50 values for E2 (Sigma Chemical Co., St Louis, Mo, USA) and puerarin (Sigma Chemical Co., St Louis, Mo, USA), i.e. the concentrations of E2 and puerarin at which half of the specific binding of [3H]-E2 had been inhibited. The relative binding affinity (RBA) was calculated as: ((IC50) E2/(IC50) puerarin) 6100%. RBA data was determined from the mean of three independent experiments, and each was performed in triplicate. Phospho-kinase Proteome Profiling ESCs were plated in 100 mm tissue culture dishes, and cultured in DMEM supplemented with 10% FBS until they reached 70% confluence. After serum starvation for 24 h, cells were then cultured in phenol red-free DMEM supplemented with 10% CSFBS with or without 1028 mol/L of E2-BSA and/or puerarin (1029 mol/L) (Sigma Chemical Co., St Louis, Mo, USA) for 20 min. Cells were then processed, following the human phospho- References 1. Bulun SE (2009) Endometriosis. N Engl J Med 360: 268–279. 2. Jacobson TZ, Duffy JM, Barlow D, Koninckx PR, Garry R (2009) Laparoscopic surgery for pelvic pain associated with endometriosis. Cochrane Database Syst Rev: CD001300. 3. Attar E, Bulun SE (2006) Aromatase inhibitors: the next generation of therapeutics for endometriosis? Fertil Steril 85: 1307–1318. Puerarin Suppresses Proliferation of ESCs kinase array kit (Proteome ProfilerTM; R&D Systems, Minneapo- lis, USA) instructions. Phospho-kinase array data were developed on X-ray films following exposure to chemiluminescent reagents. Western Blot Analysis ESCs were seeded in 40 mm dishes at a density of 36106 cells per dish. After serum starvation for 24 h, cells were treated with various doses of E2-BSA (Sigma Chemical Co., St Louis, Mo, USA ) (10210 to 1026 mol/L) or dimethyl sulfoxide (DMSO) for 15 min, or by E2-BSA (1028 mol/L) for various times as indicated. In addition, cells were exposed to 1029 mol/L of puerarin with or without E2-BSA (1028 mol/L). When inhibitors were applied, cells were pre-incubated with the ER antagonist, ICI182,780 (Sigma Chemical Co., St Louis, Mo, USA) (1 mmol/L) for 30 min, or with U0126 (Cell Signaling Technology, Beverly, Massachusetts, USA) (20 mmol/L) for 1 h. Cells were lysed, and western blotted as previously described [38]. Data S1 lists the antibodies used in this research. Cell Proliferation For cell proliferation studies, ESCs were seeded in 96-well plates (16104 cells/well) and maintained in conditions as described above. After serum starvation, the cells were further treated with the indicated compounds. The proliferative potential of cells was analyzed according to the protocol of CCK-8 (Dojindo, Japan). Reverse Transcription Reaction and Quantitative Real- time RT-PCR The ESCs (16106 cells per well) were cultured in 40 mm dishes and treated as described previously. After 24 h culture with 1029 mol/L of puerarin with or without E2-BSA (1028 mol/L), and in the presence or absence of U0126 (20 mmol/L) for 1 h, the total cell RNA in each culture was extracted and quantitative real- time RT-PCR was performed as described previously [38]. Primer sequences are shown in data S1. All samples were examined in triplicate and included no-template controls. Statistical Analysis Data were first assessed by tests of normality and homogeneity of variances, and then were represented as means 6 SD, calculated from three independent experiments. Statistical com- parisons between groups were performed using one-way ANOVA followed by post-hoc multiple comparisons where appropriate. A P value ,0.05 was considered to indicate statistical significance. Antibodies and Primers used in this research. Supporting Information Data S1 (DOCX) Author Contributions Conceived and designed the experiments: CY ZC. Performed the experiments: WC LC DZ. Analyzed the data: SY JH. Contributed reagents/materials/analysis tools: DZ JH. Wrote the paper: ZC JN CY. 4. Lessey BA (2000) Medical management of endometriosis and infertility. Fertil Steril 73: 1089–1096. 5. Wang D, Liu Y, Han J, Zai D, Ji M, et al. (2011) Puerarin suppresses invasion and vascularization of endometriosis tissue stimulated by 17beta-estradiol. PLoS One 6: e25011. PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45529