S. Sesselmann, M. D. , S. Söder, M. D. , R. Voigt, J. Haag, Ph. D. , S

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DNA methylation is not responsible for p21WAF1/CIP1 down-regulation in osteoarthritic chondrocytes S. Sesselmann, M.D., S. Söder, M.D., R. Voigt, J. Haag, Ph.D., S.P. Grogan, Ph.D., T. Aigner, M.D., D.Sc. Osteoarthritis and Cartilage Volume 17, Issue 4, Pages 507-512 (April 2009) DOI: 10.1016/j.joca.2008.09.006 Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 General methylation level of genomic DNA derived from normal and OA cartilage measured by HPLC (a). Representative absorption curve of a single sample of digested DNA as compared to a standard curve of 10mM of dC and 5mdC (b). Methylation of the individual samples shown relative to the age of the donors (c). Osteoarthritis and Cartilage 2009 17, 507-512DOI: (10.1016/j.joca.2008.09.006) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 qPCR-analysis of the expression of the DNMTs, DNMT1, DNMT3a and DNMT3b using real-time PCR (n(normal)=8, n(late-osteoarthritic)=9). Shown are values normalized to GAPDH. Osteoarthritis and Cartilage 2009 17, 507-512DOI: (10.1016/j.joca.2008.09.006) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Analysis of the expression of p21WAF1/CIP1 mRNA by conventional (a: lane 1: standard; lanes 2–4: normal samples; lanes 5–9: late OA samples; lane 10: Hela cell line; lane 11: water (negative control)) and real-time PCR (b: n(normal)=10, n(late-osteoarthritic)=10; shown are values normalized to GAPDH). Osteoarthritis and Cartilage 2009 17, 507-512DOI: (10.1016/j.joca.2008.09.006) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Analysis of p21 promoter methylation by bisulfite genomic sequencing. (a) Analysis of the CpG island in the p21 promoter region. (b) A region containing 64 CpG sites was chosen for analysis. Arrows delineate the PCR product used for sequencing, individual CpG sites are indicated by circles. (c) Ten individual alleles were analyzed for each cartilage sample (three normal, six OA). Individual alleles are arranged in rows. Filled circles depict methylated CpG sites, open circles represent unmethylated sites. The percentage of methylated CpG sites is shown next to each sample. “Conversion rate” shows the percentage of non-CpG site cytosine residues converted after bisulfite treatment. Osteoarthritis and Cartilage 2009 17, 507-512DOI: (10.1016/j.joca.2008.09.006) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions