Ye et al. BMC Cancer 2010, 10:611 http://www.biomedcentral.com/1471-2407/10/611 Page 7 of 11 Figure 2 Effects of Plexin-B1 siRNA on SKOV3 cells. (A) Real-time PCR was performed to detect Plexin-B1 mRNA levels in non-transfected cells (Blank control) and cells at 24 h after a 5 h exposure to negative control siRNA or one of the three different Plexin-B1 siRNAs (Plexin-B1 siRNA1-3). The mean relative level ± SD of each group is shown. Plexin-B1 mRNA levels in three Plexin-B1 siRNA groups were significantly down- regulated (*P < 0.01), while the negative control siRNA did not cause an obvious change. The bar graph shows the results of three independent experiments. (B) Western blot analysis of lysates from untransfected (Blank control) SKOV3 cells and SKOV3 cells at 24 h after a 5-h exposure to one of the three different Plexin-B1 siRNAs (Plexin-B1 siRNA1-3) or to negative control siRNA. The cells were analyzed by immunoblotting with specific antibodies to Plexin-B1, p-AKT (s473), AKT and b-actin. Plexin-B1 expression was significantly down-regulated in each of the three Plexin- B1 siRNA groups relative to the blank control group or the negative control group. p-AKT (s473) expression sequentially decreased in the three Plexin-B1 siRNA groups, and AKT changed impalpably at the protein level. b-actin was the internal loading control. (C) The western blots were scanned and quantified. Data present densitometric analyses of Plexin-B1, p-AKT (s473) or AKT relative to b-actin for n = 5 independent experiments. * indicates P < 0.01 when compared to the blank control. 85% and 100%, suggesting that inhibition of Plexin-B1 decreased the migration of SKOV3 cells. As shown in Figure 3D-F, the number of SKOV3 cells that passed through the filter in the Plexin-B1 siRNA2- (100 ± 52) and siRNA3- (42 ± 36) treated groups was remarkably less than that in the untreated (825 ± 32) or treated with negative control siRNA (848 ± 41) groups, which shows that inhibition of Plexin-B1 expression sup- pressed SKOV3 cell invasion in vitro. Silencing of Plexin-B1 altered the cytoskeleton of SKOV3 cells A previous study proved that Plexin-B1 regulates the actin cytoskeleton by activating Rho activity [22]. To investigate how changes in Plexin-B1 expression affect the reorganization of the cytoskeleton, rhodamine-phal- loidin was used to label F-actin in SKOV3 cells 24 h after a 5-h exposure to Plexin-B1 siRNA2 or siRNA3. As shown in Figure 4, SKOV3 cells in the transfected groups shrank without F-actin polymerization or filopo- dia formation, whereas cells in the blank and control groups showed pointed filopodia or polygonal cell shapes with pointed edges, in which brightly stained longitudinal actin bundles were detected [39]. Discussion Plexins are a family of transmembrane receptors that mediate repulsive neural responses induced by sema- phorins. Increasing evidence suggests that plexins have functions in multiple types of tumors and multiple developmental processes, such as cell adhesion, migra- tion, and apoptosis. Plexin-B1, a high-affinity receptor for Sema 4D, is widely expressed in tissues outside the ner- vous system and regulates angiogenesis [37,40], oncogen- esis [27], cell mobility [26,41], mouse ovary follicular growth and hormonogenesis [4] by initiating different signaling pathways via its cytoplasmic region, which binds and activates various intracellular molecules. It has been reported that both the expression and roles of Plexin-B1 are closely associated with cellular types and with intracellular signaling molecules such as the recep- tor tyrosine kinases ErbB2 and MET [26]. However, the biological effects of Plexin-B1 are poorly understood. Valente et al. [30] recently assessed the potential value of Plexin-B1 expression, either alone or in conjunction with Met, as a marker of tumor progression by analyz- ing their expression in a series of 50 malignant epithelial tumors (35 breast and 15 ovary). Plexin-B1 expression was found in 7/11 ovarian serous adenocarcinomas, and 6/15 ovarian adenocarcinomas expressed both Met and Plexin-B1, which was predictive of an unfavorable out- come. In the present study, immunohistochemical analy- sis was used to investigate Plexin-B1 expression in ovarian serous tumors in a larger series. Consistent with data reported previously by Valente et al., our results revealed that elevated expression of Plexin-B1 was asso- ciated with increasing tumor malignancy. Plexin-B1 levels ranged from 15.00% in normal and benign ovarian tissues through 35.00% in borderline tumors to 55.00% in malignant ovarian tumors, suggesting that Plexin-B1