, Vorinostat The Netherlands) using REDTaq® ReadyMix™ PCR Reaction mix (Sigma-Aldrich, Dorset, UK). Cycling conditions were as followed: 94°C for 5 min, 94°C for 30 s, 55°C for 30 s, 72°C for 30 s and the final extension phase at 72°C for 7 min for 36 cycles. The PCR products were CRT0066101 supplier separated on a 2% agarose gel and electrophoretically separated. The gel was

then stained with ethidium bromide prior to examine under ultraviolet light and photographs taken. Table 1 Primer sequences used in this study Expression product Primer name Expression primer sequence (5′-3′) Predicted size (bp) Claudin-5 CL5expR1 GACGTAGTTCTTCTTGTCGT 547 CL5expF2 ATGGGGTCCGCAGCGTTGGAGATCCT CL5 ribozyme1 CL5ribF1 ACTAGTCCGCAGCGTTGGAGATTTCGTCCTCACGGACT 99 Apoptosis inhibitor CL5ribR1 CTGCAGACAGCACCAGGCCCAGCTGATGAGTCCGTGAGGA CL5 ribozyme2 CL5ribF2 CTGCAGCAGGTGGTCTGCGCCGTCACCTGATGAGTCCGTGAGGA 102 CL5ribR2 ACTAGTGACCGCCTTCCTGGACCACAACATTTCGTCCTCACGGACT

β-actin BACTF ACTGAACCTGACCGTACA 580   BACTR GGACCTGACTGACTACCTCA   Real-time quantitative Polymerase Chain Reaction (Q-PCR) The assay was based on the Amplifluor system. It was used to detect and quantify transcript copy number of Claudin-5 in tumour and background samples. Primers were designed by Beacon Designer software, which included complementary sequence to universal Z probe (Intergen, Inc.). Each reaction contains 1 pmol reverse primer (which has the Z sequence), 10 pmol of FAM-tagged universal Z probe (Intergen, Inc.) and cDNA (equivalent to 50 ng RNA) (primer sequences are shown in Table 1). Sample cDNA was amplified and quantified over a large number of shorter cycles using an iCyclerIQ thermal cycler and detection software (BioRad laboratories, Hammelhempstead,

UK) under the following conditions: an initial 5 minute 94°C period followed by 60 cycles of 94°C for 10 seconds, 55°C for 15 seconds and 72°C for 20 seconds. Detection of GAPDH copy number within these samples was later used to allow further standardisation and normalisation of the samples. SDS-PAGE, Western blotting and co-immunoprecipitation MDA-MB-231 cells were grow to confluence, detached and lysed in HCMF buffer containing Oxymatrine 0.5% SDS, 0.5% Triton X-100, 2 Mm CaCl2, 100 μg/ml phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin and 10 Mm sodium orthovanadate for 1 hour, sample buffer was added and the protein boiled at 100°C for 5 min before being spun at 13,000 g for 10 min to remove insolubles. Protein concentration was quantified using Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hertfordshire, UK). Equal amounts of protein from each cell sample were added onto a 10% or 15% (depending on protein size) acrylamide gel and being subjected to electrophoretic separation. The proteins were transferred onto nitrocellulose membranes which were blocked and probed with specific primary antibodies (1:500), following with peroxidase-conjugated secondary antibody (1:1000).