Fig. 7.

DNA methylation influences human Runx2 expression in patients with steroid-associated ONFH. a DNA methylation status of human Runx2 promoter in normal tissues and necrotic tissues in patients with steroid-associated ONFH using sodium bisulfite sequencing. Each PCR product was subcloned and subjected to nucleotide sequencing analysis. Ten representatives of sequenced clones were depicted by filled (methylated) and open (unmethylated) circles for each CpG site. b–d The expression levels of human Runx2, OPG and RANKL were detected by quantitative RT-PCR. β-actin was used as loading control. The experiments were repeated three times (n = 16). e Detection of human Runx2 expression in human bone samples by immunohistochemical staining. Bone samples of NT and ONT were decalcified and sectioned. Anti-Runx2 antibody was used for immunohistochemical staining. f The protein expression levels of human Runx2 in necrotic tissues and normal tissues were detected by western blot with indicated antibodies. NT, normal tissue; ONT, osteonecrotic tissue. g Quantification of the band intensity using ImageJ software. Data are presented as mean ± SD (n = 3, *p < 0.05)