ATDC5 cells were cultured in media consisting of 47.5% Dulbecco's modified essential medium, 47.5% F12, 5% foetal bovine serum, 0.25% L-glutamine and 0.25% Penstrep (Invitrogen, Burlington, Ontario, Canada), and induced to differentiate with 1% ITS (Sigma, Oakville, Ontario, Canada), as described previously [29, 30]. Cells for RNA isolation were plated in six-well plates (Falcon) at a density of 60,000 cells/well.
For primary chondrocyte cultures, embryos were dissected from CD1 timed-pregnant mice (Charles River Laboratories, St. Constant, Quebec, Canada) at embryological day 15.5, and chondrocytes were isolated from long bones, as described previously [31, 32], by sequential digestion with trypsin and collagenase. Cells were re-suspended in media consisting of 55% F12, 35% Dulbecco's modified essential medium and 10% foetal bovine serum, and supplemented with 0.25% L-glutamine and 0.25% Penstrep (Invitrogen). The primary chondrocytes were then plated according to the desired use. For RNA and protein isolation, chondrocytes were plated in six-well plates (Falcon) at 500,000 cells per well. Primary chondrocyte cultures were treated with 1 μmol/l or 10 μmol/l of the Src family kinase inhibitor PP2 (Calbiochem; VWR, Mississauga, Ontario, Canada) or with dimethyl sulphoxide (DMSO; Sigma), the vehicle in which PP2 was dissolved, as control. For other experiments, primary chondrocytes were cultured as described above and treated with 10 μmol/l Y27632, 1 μmol/l cytochalasin D, 50 nmol/l jasplakinolide, or the vehicle DMSO.
Taqman real-time PCR
Total RNA was harvested from treated cultures on days 3, 6, 9, 12, 15, and 18 of ATDC5 differentiation, as well as from primary chondrocyte cultures treated with DMSO, PP2, cytochalasin D, Y27632, or jasplakinolide. RNA was isolated using a Qiagen (Mississauga, Ontario, Canada) RNeasy kit, following the manufacturer's instructions. RNA samples were diluted to a final concentration of 25 ng/μl for use in real-time RT-PCR, as described previously . Relative gene expression was measured using Assays on Demand (Applied Biosystems) for Col2a1, Col10a1, Agc1, Sox6, Sox5, Sox9, Chst3, Chst11, Xylt1, Xylt2, Ihh, Cdkn1c, Atf3, Lyn, Frk, and Hck in relation to the glyceraldehyde-3-phosphate dehydrogenase gene (Gapdh; Applied Biosystems, Foster City, CA, USA) using One Step RT qPCR Master Mix Plus (Eurogentec North America, San Diego, CA, USA) and 40 cycles on the ABI Prism 7900 HT sequence detector (PerkinElmer, Emeryville, CA, USA). Real-time analysis was performed as quadruplicate reactions on at least three independent trials in each experimental situation.
Protein was harvested from primary cell cultures using RIPA lysis buffer (150 nmol/l NaCl, 50 mmol/l Tris-HCl [pH 7.5], 1% Triton-X, 1% deoxycholate, 0.1% SDS, 2 mmol/l EDTA) supplemented with a protease inhibitor mini complete tablet (Roche Applied Science, Laval, Quebec, Canada). The isolated protein was then sonicated and quantified with the Bicinchoninic Acid kit (Sigma), in accordance with the manufacturer's protocol. An equal quantity (20 μg) of each sample was used in SDS-PAGE and transferred to nitrocellulose membrane (BioRad Labratories, Mississauga, Ontario, Canada). Membranes were then blocked in 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.01% Tween-20 (TBST), followed by overnight incubation at 4°C with primary antibody against Lyn (Cell Signaling Technology, Inc., Mississauga, Ontario, Canada) or β-actin (Sigma) diluted 1:200 in 5% bovine serum albumin-TBST. Blots were then washed three times for 5 minutes in TBST, followed by incubation with the appropriate HRP (horseradish peroxidase)-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hour at 4°C. Membranes were then washed three times for 5 minutes in TBST before detection of proteins by ECL Western Blot detection reagents (Amersham Bioscience, Oakville, Ontario, Canada), in accordance with the manufacturer's instructions. Banding on the membranes was visualized using a ChemiImager 5500 (AlphaInnotech Inc., San Leandro, CA, USA). Western blotting was performed on samples from three independent trials.
Cellular proliferation was determined using an MTT Cell Proliferation Kit I (Roche Applied Science), which had been tested and confirmed as a valid method for quantifying chondrocyte cell number in our laboratory [29, 34]. Primary chondrocytes were plated on 96-well plates at a density of 10,000 cells/well and treated with 1 μmol/l or 10 μmol/l of the tyrosine kinase inhibitor Tyr A23, Tyr A25, or Tyr A47, or the Src family kinase inhibitor PP2, or with DMSO alone as the vehicle control. In accordance with the manufacturer's directions, cells were treated with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) at 24, 48 and 72 hours and, following incubation and solubilization, absorbance was measured at 600 nm with a spectrophotometer as described previously . MTT assays were performed in four independent trials.
Primary chondrocytes were plated on glass coverslips in a 24-well plate at a density of 50,000 cells/well and cells were allowed to adhere overnight. The following morning, cells were treated either with 10 μmol/l PP2 or DMSO as a control and incubated for an additional 24 hours. The next day, cells were freshly treated with the inhibitor or vehicle alone. After a 1-hour incubation period, cells were washed with room temperature phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 10 minutes. After rinsing with fresh PBS, cells were incubated with 0.1% Triton-X solution in PBS for 5 minutes to allow for membrane permeability. The coverslips were then rinsed in PBS and incubated in blocking solution (1:20 goat serum [Sigma]:PBS) for 30 minutes at room temperature. After blocking, primary antibody against FAK (BioSource International, Inc.) or phospho-FAK [pY397] (BioSource International, Inc., Montreal, Quebec, Canada; 1:100 antibody:blocking solution) was added to the coverslips and incubated for 45 minutes at room temperature. After washing with PBS, the coverslips were incubated for 45 minutes in the dark at room temperature with a fluorescein isothionate-conjugated secondary antibody diluted 1,000 times in PBS. Coverslips were again washed before incubation with rhodamine-phalloidin solution (Cytoskeleton, Denver, CO, USA) for 45 minutes in a dark environment. Following incubation, the coverslips were rinsed and mounted using VectaShield with DAPI (Vector Laboratories Inc., Burlingane, CA, USA). Images of these cells were taken with a Leica DMRA2 fluorescence microscope (Quorum Technologies, Guelph, Ontario, Canada) at 63-fold magnification and analyzed using OpenLab software. (Quorum Technologies, Guelph, Ontario, Canada). Fluorescence imaging was performed on at least three independent trials.
RhoA activity assay
Primary chondrocytes were cultured with DMSO or PP2 for 1 to 3 days. Ras homology A (RhoA) activity was measured using a luminescent G-LISA™ kit (Cytoskeleton), as described previously .
All experiments were performed in at least three independent trials. Real-time PCR data represent an average of three independent trials of samples run in quadruplicate, normalized to Gapdh and to day control (DMSO) levels. Statistical significance was determined using a one-way analysis of variance with Bonferroni's post-test (P < 0.05). Statistical tests were performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA).