Yu et al. Journal of Experimental & Clinical Cancer Research 2010, 29:119 http://www.jeccr.com/content/29/1/119 Page 3 of 9 (BioGenex) for 5 min. The primary antibodies for Snail, Slug, Twist, and E-cadherin were either polyclonal rabbit anti-Twist and anti-E-cadherin or polyclonal goat anti- Snail and anti-Slug, and purchased from Santa Cruz Biotechnology. Antibody dilution ranged from 1:50 to 1:150 in PBS for 30 min at 37°C. As negative control, sections were incubated with PBS instead of the primary antibody. This was followed by incubation with biotiny- lated antirabbit/antigoat immunoglobulin G (1:200; Santa Cruz Biotechnology) for 30 min at 37°C and per- oxidase-conjugated avidin-biotin complexes (KPL) and 3,3′-diaminobenzidine (Sigma). The sections were then counterstained with Mayer’s hematoxylin, upgraded alcohols, mounted, and analyzed by standard light microscopy. Evaluation of immunohistochemistry results Immunohistochemical staining of Snail, Slug and Twist and E-cadherin was defined as detectable immunoreac- tion in perinuclear and/or cytoplasm. Expression of Snail, Slug and Twist was considered negative when no or less than 49% of the tumour cells were stained[16]. Cancer cells that were immunostained less than 10% staining were defined as having a reduced E-cadherin expression[17]. Cell lines The human bladder cancer cell lines (T24, HTB-3, HTB-1, HTB-2 and HTB-9) obtained from ATCC (Rockville, MD, USA). All the cell lines were maintained in RPMI 1640 medium supplemented with 10% FCS, 1% penicillin and streptomycin at 37°C in 5% CO2. Reverse transcription-polymerase chain reaction Total RNA from the cell lines were obtained using RNeasy Mini kit(Qiagen, Tokyo, Japan) according to the manufacture’s instructions and resuspended in 50 μL dimethylpyrocarbonate-treated water. RNA concentra- tion was determined using a BioPhotometer (Eppendorf Scientific). Total RNA (2 μg) was primed with an oligo (dT) oligonucleotide and reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Promega) and deoxynucleotide triphosphates (Sigma- Aldrich) according to the instructions of the manufacturer. First-strand cDNA was amplified with transcript-specific oligonucleotides using Ready-Mix Taq PCR Reaction Mix (Sigma-Aldrich). The primers (TIB Molbiol) for the respective genes were designed as fol- lows: Slug (533 bp) 5′-GGTCAAGAAGCATTTCAAC-3′ (sense) and 5′-GGTAATGTGTGGGTCCGA-3′ (anti- sense);Snail (557 bp) 5′-CAACCCACTCAGATGTCAA- 3′ (sense) and 5′-CATAGTT AGTCACACCTCGT-3′ (antisense); Twist (527 bp) 5′-GGGAGTCCGCAGTCT- TAC-3′ (sense)and5′-CCTGTCTCGCTTTCTCTTT-3′ (antisense); E-cadherin (420 bp)5′-ATTC TGAT TCTGCTGCTCTTG-3′ (sense)and 5′-AGTAGTCATAG TCCTGGTCTT-3′(antisense);and b-actin (335 bp) 5′- TTCCTGGGCATGGAGTCCTGTGG-3′ (sense) and 5′- CGCCTAGAAGCATTTGCGGTGG-3′ (antisense). The condition of PCR for Slug were: initial denaturing at 95° C for 10 min, followed by 38 cycles of denaturing at 94° C for 60 s, annealing at 53°C for 60 s and extension at 72°C for 90 s. All PCR products were visualized by elec- trophoresis and ethidium bromide staining in 2% agar- ose gels. RT-PCR was performed in a triplicate. Western blotting analysis For isolation of total protein, cells were washed twice with ice-cold PBS containing phosphatase inhibitor cocktail II (Sigma-Aldrich), scraped of the culture flask, pelleted by centrifugation, and lysed in buffer containing 10 mmol/L Tris (pH 6.8), 2 mmol/L EDTA (pH 8.0), 0.15 mol/L NaCL, 0.1% Brij 96, 0.1% NP40, 2 mmol/L phenylmethylsulfonyl fluoride, and 1× Protease inhibitor cocktail (Sigma-Aldrich). Protein was estimated using QuantiPro bicinchoninic acid assay kit (Sigma-Aldrich) according to the instructions of the manufacturer[16]. Ten micrograms of proteins were denatured at 95°C with sample buffer [0.125 mol/L Tris (pH 6.8), 4% SDS, 20% glycerol, 2% mercaptoethanol, 0.03 mmol/L brom- phenol blue] for 5 min and separated by electrophoresis in 7.5% to 12% SDS-PAGE gels according to their mole- cular weight. Proteins were transferred onto a polyviny- lidene difluoride membrane (Perkin-Elmer) and blocked for 2 h in blocking solution (5% nonfat dry milk in TBS containing 0.1% Tween 20) followed by 5% bovine serum albumin in TBS/Tween at room temperature on a rotating plate for 2 h. The membrane was then exposed to the primary antibody overnight at 4°C. The primary antibodies were the same we used for immuno- histochemistry, and the dilution was 1:200 in Snail, Slug, Twist, and E-cadherin, and 1:500 in b-actin. After wash- ing, the membranes were incubated for 1.5 h at room temperature with peroxidase-linked secondary antibody (Roche), and signals were detected using Lumilight Plus Western blotting kit reagents (Roche) according to the manufacturer’s instructions and luminescence imaging (LAS-1000, Fujifilm). Statistical analysis We used the c2 and Fisher’s exact tests to evaluate the differences of staining of E-cadherin and Snail, Slug and Twist according to patient and cancer characteristics. The overall survival was defined as the time between the date of surgery and the last date of follow-up or date to death owing to bladder cancer. The progression- free survival was defined as the time interval between the date of surgery and the date of progression