Figure 4.

Analysis of the regulation of SOD2 in human articular chondrocytes (HAC) by DNA methylation. A, Primary HAC were treated with the DNA demethylating agent 5-aza-2′deoxycytidine (AZA) for 35 days (2 passages) as described and SOD2 expression measured by real-time RT-PCR and normalised to GAPDH expression levels. Data is presented fold over control and error bars represent SEM. Data is combined from four HAC populations treated in duplicate. Statistical significance was calculated using the Mann-Whitney U-test, where *** represents P<0.001. B, DNA methylation analysis of the SOD2 promoter in cartilage genomic DNA from neck of femur (NOF) and osteoarthritis (OA) patients. Genomic DNA from cartilage of 12 NOF and 12 OA patients was analysed to determine the methylation status of the SOD2 promoter region spanning 19 potential CpG dinucleotides. An average of 9.6 non-clonal DNA sequences per patient sample were analysed and the methylation percentage for each is plotted. Methylation CpG sequences are represented by black bars, open are non-methylated and the bar is grey when the CpG sequence was not detected, generally due to a SNP at that locus (e.g. CpG10). Fisher’s exact test was used for statistical analysis, where * represents P<0.05 and ** P<0.01.