1mgml?1 phenylmethylsulfonyl fluoride. Re-suspended cells were passed through the 21-gauge needle to shear the DNA and incubated for 60min on ice followed selleckchem DZNeP by centrifugation at 10000g for 10min at 4��C. Total protein (10��g) was analysed by western blotting using primary antibodies and anti-mouse and anti-rabbit IgG HRP-conjugated secondary antibodies (Dako, Glostrup, Denmark), and were visualised with LumiGLO Reagent and Peroxide (Cell Signaling Technology, Beverly, MA, USA). Results of western blot analysis were shown as ratio of band intensity, which indicates the ratio of band intensity of tested protein to that of ��-actin. Band intensity was measured by using ImageJ software (NIH, Bethesda, MD, USA). At 48h after transfection, cells were assayed for Rho activation with a Rho Activation Assay Kit (Upstate).

The GTP-bound fraction was monitored by western blot analysis. The primary antibodies used were mouse anti-V5 (Invitrogen), anti-��-actin (Abcam, Cambridge, UK), anti-p38, anti-phospho-p38, anti-ERK, anti-phospho-ERK, anti-JNK, anti-phospho-JNK, anti-c-Jun and anti-phosphor-c-Jun (BD Biosciences, San Jose, CA, USA) monoclonal antibodies, and rabbit anti-RhoA monoclonal antibody (Cell Signaling Technology). Cell invasion assay Matrigel (1:5; BD Biosciences) was added to Transwell membrane filter inserts (8.0��m pore size; Costar, Cambridge, MA, USA) and incubated for 5h at 37��C in a 5% CO2 tissue culture incubator. HCT-116 cells were transfected with scramble siRNA or FZD7_siRNA8. At 24h after transfection, cells were harvested and re-suspended in serum-free medium.

Aliquots (105 cells) of the prepared cell suspension were added into the upper chamber and the lower chamber was filled with 600��l of culture media containing 5��gml?1 fibronectin (Sigma), as an adhesive substrate. Cells were incubated for 48h at 37��C in a 5% CO2 tissue culture incubator. Invasive cells were stained with Diff-Quick solution (Fisher Scientific, Pittsburgh, PA, USA). Cells were counted with a microscope. The average number of cells in five fields per membrane was counted in triplicate. Quantitative PCR Total RNA was isolated from colon cancer cell lines and primary colorectal tumour and non-tumour tissues using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) and All prep DNA/RNA Mini kit (Qiagen), respectively.

The extracted total RNA was reverse-transcribed into single-stranded cDNA using a High-Capacity Carfilzomib cDNA Archive Kit (Applied Biosystems, Warrington, UK). Real-time PCR was performed using first-strand cDNA with TaqMan Universal PCR Master Mix (Applied Biosystems). The assay numbers for the endogenous control (��-actin) and target genes were as follows: 4326315E (��-actin); Hs00275833_s1 (FZD7). Quantitative PCR was performed on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Quantitative PCR parameters for cycling were as follows: 50��C for 2min hold, 95��C for 10min 40 cycles of PCR at 95��C for 15s and 60��C for 1min.