Ye et al. BMC Cancer 2010, 10:611 http://www.biomedcentral.com/1471-2407/10/611 Page 4 of 11 FBS. Then, 20 μl of MTT (5 mg/ml, Sigma Chemical Co.) was added to each well and incubated for 4 h at 37°C, which was followed by adding 100 μl DMSO and incubating at 37°C for an additional 20 min. Absorbance was read at 570 nm on a microplate reader. Statistical analysis All experiments were repeated at least three times. The software package SPSS13.5 was used to analyze data. P < 0.05 was defined as statistically significant. All values were presented as means ± standard deviation (SD). Wound-healing assay Cells were seeded in individual wells of a 6-well culture plate. Untransfected cells and cells exposed to Plexin-B1 siRNA or negative control siRNA for 5 h were cultured in quiescent medium for 24 h. Then, a sterile 10-ul pip- ette tip was used to longitudinally scratch a constant-dia- meter stripe in the confluent monolayer. The medium and cell debris were aspirated away and replaced with 2 ml of fresh serum-free medium. Photographs were taken at 0 h, 24 h and 48 h after wounding (corresponding to 24 h, 48 h and 72 h post-transfection) by phase contrast microscopy. For statistical analysis, three randomly selected points along each wound were marked, and the horizontal distance between the migrating cells and the initial wound was measured at 24 h and 48 h. Values were means ± SD from at least three-independent experi- ments. Differences between siRNA-treated and blank control data were determined by a Student’s t-test, where P < 0.05 was considered significant. Cell invasion assay Cell invasion was assayed using Transwell chambers (Costar, Cambridge, MA, USA) with 8-μm pore polycar- bonate filters that were coated with Matrigel™ (BD Bios- ciences, Franklin Lakes, NJ). Cells cultured in the absence or presence of siRNA for 24 h were seeded into the upper chambers in serum-free medium at a density of 2.5 × 104 per well, and 500 μl of NIH3T3 conditioned medium was placed in the lower chamber as a chemo- attractant. After 48 h at 37°C in 5% CO2, the cells were fixed with 3.7% paraformaldehyde and stained with 0.1% crystal violet solution. Cells on the upper surface of the filter were removed with cotton buds. Invaded cells on the underside of the filter were photographed and counted by phase contrast microscopy (×200 magnifica- tions). The experiments were performed in triplicate. Rhodamine phalloidin SKOV3 cells were plated overnight on sterile glass cov- erslips in a six-well plate 24 h after a 5-h exposure to Plexin-B1 siRNA, negative control siRNA, or no treat- ment. The cells were washed in PBS, fixed in 3.7% par- aformaldehyde, permeabilized in 0.1% Triton-X100, incubated in blocking solution (1% bovine serum albu- min [BSA]), and then were kept for 20 min at room temperature in rhodamine-phalloidin solution. Images were acquired with a confocal laser scanning microscope (The Olympus FluoView FV1000). Results Expression of Plexin-B1 in human ovarian tissues with different pathological changes To elucidate the expression of Plexin-B1 protein during serous ovarian carcinogenesis and to study the associa- tion of Plexin-B1 expression with clinical-pathological parameters, the expression levels of this protein in nor- mal human ovaries, benign ovarian tissue, serous border- line ovarian tumors and malignant ovarian tumors were investigated with immunohistochemistry. As summarized in Table 1, Plexin-B1 immunoreactivity was present in 3 (15.00%) normal and benign ovarian samples (Figure 1A, 1B), in 7 (35.00%) borderline tumors (Figure 1C) and in 44 (55.00%) cystadenocarcinomas. The expression of Plexin-B1 in serous ovarian cystadenocarcinomas was statistically higher than that in the normal and benign tis- sues. In borderline tumors, the expression levels of Plexin-B1 were neither significantly higher nor signifi- cantly lower than the levels in the normal and benign tis- sues or the cystadenocarcinomas. Furthermore, in ovarian cystadenocarcinomas, the membrane and cyto- plasm of the tumor cells was positive for Plexin-B1 immunostaining (Figure 1D), and a significant difference (P = 0.008) in positive staining was observed between samples with lymph node metastasis (75.00%) and those without lymph node metastasis (44.23%). Although Plexin-B1 immunoreactivity increased gradually from stage I (45.00%) to stage IV (70.00%), there were no sta- tistical differences. No correlation was found between Plexin-B1 expression and age or cell differentiation. Expression of Plexin-B1 in human ovarian cancer cell lines To further investigate Plexin-B1 expression in ovarian cancer cells, RT-PCR and western blot analysis were used to detect Plexin-B1 mRNA and protein levels in four human ovarian cancer cell lines: SKOV3, A2780, C13* and OV2008. The results showed that Plexin-B1 expression levels were the highest in the SKOV3, whereas faint Plexin-B1 expression was found in the OV2008 cells (Figure 1E, F). Thus, SKOV3 cells were selected as a model for further investigation. Specific silencing of Plexin-B1 in SKOV3 cells with Plexin- B1 siRNA The experiments described in this study were performed by transfection of SKOV3 cells with 200 pmol siRNA for 24 h, which offered the best silencing efficiency in our preliminary experiments.