Skin fibroblasts were obtained from the original proband and an age-matched healthy counterpart, as previously described . Written informed consent for participation in the study was obtained from the parents of the proband. Furthermore, the use of the cells for this study was in accordance with the regulations of the ethics committee of the University of Portsmouth. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% foetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (Lonza, Slough, UK). Cells were maintained by incubation at 37°C in a humidified atmosphere of 5% CO2 in air and passaged sparingly once a week.
Real-time quantitative reverse transcription-PCR (qRT-PCR)
Human TNS3 gene expression in the fibroblasts was analysed by quantitative RT-PCR (qRT-PCR) using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Warrington, UK). Different sets of fluorescent primers and probes for TNS3 and a housekeeping gene (GAPDH) were used for multiplex real-time PCR detection of expression (Integrated DNA Technologies, Leuven, Belgium). Total RNA was first extracted from the fibroblasts using a kit (Qiagen, Crawley, UK), and subsequently reverse transcribed in a reaction mix containing oligodT primers, dNTPs, RT buffer, RNase inhibitor and reverse transcriptase (Fermentas, St. Leon-Rot, Germany). The resulting cDNA (5 μl) was then added to a qPCR reaction mix containing TNS3 and GAPDH primers and probes, and qPCR polymerase master mix (Maxima, Fermentas) to a final volume of 20 μl. The cycling conditions were as described previously . Each measurement was performed in triplicate wells per reaction. The relative standard curve method of quantitation was employed, using a standard curve of TNS3 expression in cDNA from the human glioma cell line SNB-19.
SDS-PAGE and western blot
Fibroblasts at near confluence in 6-well tissue culture plates were rinsed in ice-cold phosphate-buffered saline (PBS) and lysed in ice-cold lysis buffer composed of 1% NP-40, 1% deoxycholate, 5 mM EDTA, 1 mM EGTA in PBS, pH 7.4, supplemented with a protease inhibitor cocktail (Calbiochem, Nottingham, UK). Cell lysates were clarified by centrifugation at 20,800 rcf for 30 min at 4°C, from which the supernatants were transferred to new tubes. Total protein concentration was measured with using a bicinchoninic acid (BCA) assay kit (Sigma, Gillingham, UK). Equal protein amounts were loaded onto 10% polyacrylamide gels for reducing SDS-PAGE, after which proteins were transferred onto a PVDF membrane. Membranes were blocked in 5% milk powder in 25 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 8.0, and then probed with primary antibodies at optimal dilutions. The anti-Tensin3 antibodies used included two in-house rabbit polyclonal antibodies, raised against the N- and C-terminal regions of Tensin3, the C-terminal antibody having been characterised previously . Also, two new commercial anti-Tensin3 polyclonal antibodies were used, raised in goat (Santa Cruz Biotechnology, CA) and rabbit (Sigma). Furthermore, a goat polyclonal ExoC6B antibody (Santa Cruz) was also tested on lysates. Membranes were incubated with the primary antibody overnight at 4°C, then washed 3 times for 5 min in wash buffer, 0.1 M Tris–HCl, 0.15 M NaCl, 0.05% Tween 20, pH 8.0. They were then incubated for 1 h at room temperature with secondary antibodies against the relevant species primary antibody, conjugated to horseradish peroxidase (HRP) (Promega, Southampton, UK). Further washes were followed by chemluminescent development using an H2O2 and luminol-based reagent mixture (HRP, Sigma).
For immunoprecipitation (IP) experiments, fibroblasts were seeded into 10-cm tissue culture dishes and grown to near confluence before lysis. Cells lysates were prepared as for SDS-PAGE described above, and protein concentration was determined so as to prepare samples containing 500 µg total protein. Before IP, lysates were initially pre-cleared by incubation with with 10 μl protein A/G-agarose beads (Alpha Diagnostics, San Antonio, TX) and mixed with gentle end-to-end rotation for 1 h at 4°C. The beads were sedimented by quick centrifugation, and the supernatant collected into separate tubes to which 3 μg IP antibody was added; tubes were rotated overnight at 4°C. The next day, the beads were washed 3 times with ice-cold lysis buffer and once with PBS. The proteins bound to beads were removed by addition of 3x SDS loading buffer and brief boiling before being subjected to 10% SDS-PAGE and western blot for Tensin3 detection, as described above.
Immunofluorescence staining and confocal microscopy
Fibroblasts were plated onto glass coverslips and allowed to adhere overnight at 37°C, 5% CO2. The next day, cells were rinsed with PBS and fixed with 4% formaldehyde for 15 min, and permeabilised with 0.2% Triton X-100 for 10 min. For staining, cells were first blocked for 1 h with 10% horse serum in PBS and then incubated with the primary antibody (rabbit anti-Tensin3 in 1.5% serum) for 2 h at room temperature. Cells were washed three times with PBS and then incubated with donkey anti-rabbit secondary antibodies conjugated to Alexa Fluor® 647, at 1:1000 dilution in 3% serum for 1 h at room temperature. The coverslips were washed three times with PBS and mounted onto glass slides using an aqueous, non-fluorescent mountant (Polysciences, Eppelheim, Germany). The samples were analysed under a confocal laser-scanning microscope (LSM710; Zeiss, Oberkochen, Germany), using a Plan Apochromatic 63× DIC oil objective (NA1.4). Images were processed with the software Zen2008 Light Edition (Zeiss). Specificity of the staining was verified by lack of fluorescence in cells incubated with secondary antibodies alone.
Furthermore, live cells in culture were also viewed under phase contrast microscopy, and images were captured using a CCD digital microscope camera (DP50, Olympus).
Scratch-wound assay and live cell imaging microscopy
Fibroblasts were seeded in equal numbers (1.5 × 104) into wells of a 24-well tissue culture plate and left to adhere overnight at 37°C and 5% CO2 prior the experiment. Cells were at 90% confluence the next day, at which point they were pre-incubated for 1 h with the live cell fluorescent dye Cell Tracker ™ Blue (Life Technologies, Paisley, UK). In each well, three straight, parallel scratches were made across the diameter of the well using a 0.58 mm orifice size pipette tip. From that point on, filling of the wound by migrating cells was monitored under a live cell (time-lapse) imaging system comprising a Zeiss Axiovert 200 M microscope (Carl Zeiss, Welwyn Garden City, UK) fitted with an incubator (37°C, 5% CO2, humid atmosphere). A 5x (air) objective was used; fluorescent images were acquired using Volocity software (v5.4; Perkin Elmer, Bucks, UK) such that 3 points across separate wound sections in each well were imaged every 1 h for 72 h to measure wound closure. The images were collated and movie sequences generated. Image analysis of cell movement was carried out using the free image processing program ImageJ (http://rsb.info.nih.gov/ij/). Experiments were performed in triplicate.
The levels of Tensin3 mRNA, the rates of cell migration and proliferation at specific time points, and cell width in control vs proband fibroblasts were compared by unpaired t test. A P < 0.05 was considered statistically significant.