Puerarin Suppresses Proliferation of ESCs Figure 4. Effect of E2-BSA and puerarin on cell proliferation in ESCs. (A) ESCs were treated with various concentrations (10210 to 1026 mol/L) of E2-BSA for 4 d. (B) Growth curves of ESCs incubated with E2-BSA (1028 mol/L) and/or puerarin (1029 mol/L) for 0, 1, 2, 4 d. (C) Cells were treated for 4 d with E2-BSA (1028 mol/L) in the absence or presence of puerarin (1029 mol/L) and/or pretreated with U0126 (20 mmol/L) for 60 min. The figures represent data obtained from three experiments; each experiment was performed in triplicate, and data are represented as the mean 6 SD. DMSO treatment was used as the vehicle control. Vs. control,**, P,0.01, *, P,0.05; vs. E2-BSA, m, P,0.05. doi:10.1371/journal.pone.0045529.g004 partly through activating ERK1/2 signaling pathway. Unexpect- edly, puerarin was shown to be able to partially rescue inhibiting effects of the kinase inhibitor. This result suggested that when ESCs was pretreated with U0126, non-genomic ERK pathway of E2-BSA and puerarin was blocked, however, genomic effects of puerarin were still remained and played a weak estrogenic role (Fig. 3C). Some literatures documented that expression of endometriosis- related genes; cyclin D1, cyp19 and cox-2 depend on non-genomic ERK pathway [16,34–35]. In addition, we found that expression of these genes, as well as phosphorylation of ERK1/2 was completely inhibited by U0126 in ESCs. This supports the conclusion that the ERK1/2 signaling pathway plays an important Figure 5. Analysis of expression of cyclin D1, cyp19 and cox-2 genes in ESCs subjected to various treatments. ESCs were treated with E2-BSA, puerarin or both for 24 h, in the absence and presence of pretreatment with U0126 for 60 min. Total RNA was isolated, RT quantitative real-time PCR was performed and relative expression of cyclin D1 (A), cyp19 (B) and cox-2 (C) were obtained. GAPDH served as an internal control. Individual bars show relative mRNA levels expressed as the mean 6 S.E.M., determined from three separate assays. Protein levels were detected by western blotting (D). Group vs. control, **, P,0.01 and *, P,0.05; Group vs. E2-BSA, mm, P,0.01 and m, P,0.05. doi:10.1371/journal.pone.0045529.g005 PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45529