Informed consent was obtained in writing from all patients who offered cartilage. Human articular cartilage was obtained from the macro scopically preserved areas within osteoarthritic knee joints during prosthetic surgery. http://www.selleckchem.com/products/Bicalutamide(Casodex).html Primary cultured human articu lar chondrocytes were prepared from those cartilages by serial enzymic digestion using Pronase and Collagenase P. Following digestion, chon drocytes were plated onto polystyrene culture dishes at a density of 2 105cm2, and maintained in Dulbeccos modified Eagles mediumF 12 containing 10% fetal bovine serum and 25 ugml ascorbic acid. For pellet culture, 1 106 chondrocytes were placed in a 1. 5 ml polyethylene centrifuge tube, which was centrifuged at 200 g for 5 minutes to form a pellet at the bottom. The pellets were maintained in the media used for the monolayer culture.

RNA interference All siRNAs were obtained from Qiagen. Sequences for these siRNAs Inhibitors,Modulators,Libraries are provided in Additional Inhibitors,Modulators,Libraries file 1. siRNAs were introduced into primary cultured chondrocytes by electroporation using a Nucleofector, following the manufacturers protocol with some modifications. For each gene, two or three siRNAs were used to suppress the expression, Inhibitors,Modulators,Libraries which was confirmed by quantitative PCR. The suppression of RRAS expression was also confirmed at the protein level by western blotting, while the suppression of expression of 5, 10, 11, v, B1, B5 and B8 integrins was validated by flow cytometry. Generation of recombinant adenoviruses Recombinant adenoviruses carrying constitutively active or dominantly negative mutants of HRAS, RRAS, RAP1A, RAP1B, and CDC42 were generated using a ViraPower Adenovisal Expression System as described before.

In brief, human HRAS, RRAS, RAP1A, RAP1B, and CDC42 complementary DNA were cloned into the adenoviral generating constructs after the introduction of CA or DN mutations. These constructs were then Inhibitors,Modulators,Libraries transfected into 293A cells using FuGENE 6, and the cells were sub cultured to generate recombinant adenoviruses carrying these genes under the control of the human cytomegalo virus immediate early enhancerpromoter. The viruses were titrated by limiting dilution plaque titration on 293A cells, and used at 50 to 100 plaque forming unitscell. In preliminary experiments, the efficiency of transduction by this method was confirmed to be almost 100%. Cell attachment assay A cell attachment assay was performed based on a previ ously described method.

In brief, primary cultured human chondrocytes were prepared and maintained in a monolayer as described earlier. For assay, the cells were harvested and suspended in serum free media Inhibitors,Modulators,Libraries at AG014699 a density of 1 106 cellsml. After a 90 minute recovery time, 100 ul cell suspension was placed in each well of a 96 well microtiter plate, some wells of which were precoated with fibronectin or BSA.