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. 2019 Aug 7;14(8):e0220653. doi: 10.1371/journal.pone.0220653

Fig 8. Increased 5-hmC occurs in osteoblast and chondrocyte gene promoters.

Fig 8

After treatment with β-glycerophosphate (BG), BG with AA, or BG with AA and Iox2, a PHD2-specific inhibitor, DNA was extracted from MC3T3-E1 pre-osteoblasts and primary rib chondrocytes. An antibody specific for 5-hmC was used to pull down DNA rich in 5-hmC, and real-time PCR with primers for CpG-rich areas of osteoblast and chondrocyte gene promoters was used to determine the 5-hmC content of those promoters as an indirect measure of their demethylation status. (A) AA treatment significantly increased 5-hmC levels in the promoters of osteoblast differentiation genes alkaline phosphatase (ALP), Indian hedgehog (Ihh), and osterix (Osx). (B) Inhibition of PHD2 with Iox2 significantly decreased 5-hmC levels in the promoters of chondrocyte genes HIF1α and VEGF. Results are presented as mean fold-change ± SEM. *P < 0.05 (Student’s t-test), n = 3 per group.