2.4. Osteogenic Induction and Inhibitor Treatment of Dental Progenitors

PDL and AB progenitors were seeded at a density of 26,000 cells/cm² and cultured overnight before treatment. Osteogenic differentiation was carried out by culturing cells with osteogenic induction medium (complete media supplemented with 50 µg/mL ascorbic acid 2-phosphate, 10 mM β-glycerophosphate, and 10 mM dexamethasone; all from MilliporeSigma). Cells were harvested after 7 or 14 days for ChIP assays, after 7 days for alkaline phosphatase assays, and after 21 days for Alizarin Red S staining assays. The induction media were changed every alternate day. Progenitor cells grown in regular culture media were used as a reference group (day 0). For epigenetic inhibitor treatment, PDL progenitors were cultured in the presence of H3K27me3 inhibitor, DZNep (MilliporeSigma) (5 µM, 10 µM, or 20 µM), and H3K9me3 inhibitor, Chaetocin (MilliporeSigma) (5 nM, 10 nM, or 20 nM), for 48 h, and the total RNA was isolated as described below.