A differentiated chondrocyte phenotype is maintained but sphingomyelinase treatment reduces type II collagen expression. Bovine articular chondrocytes were cultured as monolayers for 7–10 days in ITS supplemented media in the presence or absence of SMase (0.1 U/ml). Where cultures were extended to 10 days, media and treatments were refreshed at day 7. (a) Equivalent numbers of cells and their associated matrix and (b) media were resolved on 7.5% (weight/vol) SDS-PAGE gels. Samples were analyzed for type II collagen by Western blotting using our monoclonal antibody (AVT6E3). In addition, cells cultured in the presence (+) or absence (-) of SMase (0.1 U/ml) for this period were placed into TRIZOL® (1 × 106 cells/ml) and total RNA extracted, in accordance with the manufacturer's instructions. (c) cDNA was generated and PCR performed using primers to type IIA and IIB procollagen and Sox9. cDNA integrity was confirmed using primers to GAPDH. (d) The relative expression level, normalized to GAPDH, of aggrecan and type II collagen mRNAs was determined by quantitative PCR. Data are presented as mean ± standard error. α1(II), α1 chain type II collagen. bp, base pairs; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ITS, insulin-transferrin-sodium selenite; RT-PCR, reverse transcription polymerase chain reaction; SMse, sphingomyelinase.
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