Ye et al. BMC Cancer 2010, 10:611 http://www.biomedcentral.com/1471-2407/10/611 considered Plexin-B1 positive according to a recent report [30]. Furthermore, invasive human ductal breast cancer samples that had been confirmed as highly expressing Plexin-B1 in our previous study [34] were used as positive controls, and negative controls were carried out by incu- bating with non-immune mouse immunoglobulin (IgG) in place of the primary antibody. Three observers who were unaware of the clinical data independently scored the sec- tions. Discrepancies in the scoring process were reviewed, and a consensus was ultimately reached. RT-PCR Total RNA was extracted from ovarian cancer cells using Trizol Reagent (Invitrogen, Life Technologies, CA, USA) following the manufacture’s protocol. cDNA was synthesized from 2 μg of total RNA using Superscript reverse transcriptase (Life Technologies, CA, USA). Pri- mer sequences used for Plexin-B1 detection were as fol- lows, sense:5’-GCAGTGTGTTATCCTTTAATGAAA-3’; and antisense:5’-CCACTACAAACTGACCCCTC-3’, producing 150-bp fragments. The housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) was used for normalization (sense: 5’-TGATGACATCAA- GAAGGTGGTGAAG-3’; and antisense: 5’-TCCTT GGAGGCCATGTGG GCCAT-3’, 240 bp product). The amplification conditions were 95°C for 1 min followed by 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 45 s with a 5 min 72°C extension. PCR products were analyzed by 1.2% agarose gel electrophoresis with ethidium bromide for UV light transilluminator visualization. Western blotting Confluent cells were lysed in lysis buffer (20 mM Tris- HCl, pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM h-glycerolphosphate, 1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mg/mL leu- peptin). Protein lysates (200 μg/lane) were subjected to 8% polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. Non-specific binding sites were blocked by incubating the nitrocellulose membrane for 1 h at 37°C with 5% nonfat dried milk in Tris-buffered saline containing 0.05% Tween-20 (TBST). Membranes were incubated overnight at 4°C with primary antibodies, then washed and incubated with horseradish peroxidase conjugated secondary antibody (Cell Signaling Technol- ogy, Danvers, MA). Bands were visualized using enhanced chemiluminescence system (ECL, Pierce, USA). siRNA preparation and cell transfection Three different siRNA duplexes targeting Plexin-B1 sequences as well as a negative control siRNA were commercially designed and synthesized by Invitrogen, Page 3 of 11 Carlsbad, CA, USA. The sequences were as follows: Plexin-B1 siRNA1, sense: 5’-UCAAAUUGCUGGCUA- CUGCAGGAGG-3’; and anti-sense: 5’-CCUCCUGCA- GUAGCCAGCAAUUUGA-3’; Plexin-B1 siRNA2, sense: 5’-AACUCAGGCAAAGAGUCAGGUGCCU-3’; and anti-sense: 5’- AGGCACCUGACUCUUUGCCUGA- GUU-3’; Plexin-B1 siRNA3, sense: 5’- AAGGUAUACA- GACAGAUGGACAUCC-3’; and anti-sense: 5’- GGA UGUCCAUCUGUCUGUAUACCUU-3’. Negative con- trol siRNA, sense: 5’-AGGAGAUAUUUCGAGGCUU-3’; antisense: 5’-AAGCUCGAAAUAUCUCCU-3’. The siRNA sequences were submitted to BLAST http://www. ncbi.nlm.nih.gov/blast/ to ensure their specificity of targeting. SKOV3 cells (3 × 105) were plated in 6-well plates and allowed to adhere for 24 h. 200 pmol siRNA and 5 μl Lipofectamine™ 2000 (Invitrogen) per well were diluted separately in serum-free Opti MEM (Invitrogen) for a final volume of 250 μl, gently mixed, and incubated at room temperature for 5 min. Then, the diluted siRNA solution and the diluted Lipofectamine2000 were mixed gently and incubated at room temperature for 20 min. The cells were washed with serum-free McCOY’s 5A, and then the diluted siRNA/Lipofectamine2000 complex was added into the 6-well plates containing 1500 μl serum-free McCOY’s 5A for 5 h, after which the com- plex was replaced with normal cell culture medium. Semi-quantitative real-time RT-PCR Total RNA was isolated with TRIzol Reagent (Invitro- gen) from cells cultured in the absence or presence of siRNA for 24 h. Real-time RT-PCR amplifications were carried out using DNase I (Promega)-treated total RNA. Reactions were performed in a Stratagene MX3000P system using Real-time PCR Master Mix (TOYOBO, Japan). Primer sequences for real-time PCR were as fol- lows, Plexin-B1: sense, 5’-ATGTCACCTGCCAGCAG- CAC-3’, antisense, 5’-CAGCACTGTCCACACGCAG A- 3’; 18S: sense, 5’-AGTCCCTGCCCTTTGACACA-3’, antisense, 5’-GATCCGAGGGCCTCACTAAAC-3’. The reaction conditions were 95°C for 1 min followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 30 s. The ΔCt method was used to obtain relative expression values of Plexin-B1 as compared with 18S gene amplification. Cell proliferation assays Cell proliferation was determined by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells cultured in the absence or presence of Plexin-B1 siRNA or negative control siRNA for 24 h were seeded into a 96-well plate at a density of 5 × 103 per well. After being starved overnight in quies- cent medium, the cells were cultured for 72 h with 10%