Fig. 5.

DNMT3B suppresses PTEN expression to promote adipogenesis and inhibit osteogenesis. DP-MSCs and BM-MSCs were separately isolated from three different individuals. a Methylation-specific PCR of PTEN. U, fragments amplified by unmethylated sequence-specific primers; M, fragments amplified by methylated sequence-specific primers. b Western blotting analysis of cells treated without or with 1 μM 5 aza-cytidine (5aza) for 4 days. c Western blotting analysis of DP-MSCs and BM-MSCs. d–h BM-MSCs were transduced with control shRNAs (CTR) or with different DNMT3B shRNAs (shDNMT3B (1) and (2)). Cells were analyzed by d methylation-specific PCR of PTEN and e western blotting. f Cells were induced for adipogenic induction for 3 weeks and stained by nile red staining. Right Quantification of nile red positive cell percentages at 3 weeks. Bar = 50 μm. g Cells were induced for osteogenic differentiation for 2 weeks and stained by Arsenazo III kit. Right Quantification of the calcium contents relative to DNA contents. Bar = 200 μm. Results are expressed as the mean ± SD of three independent experiments. h DP-MSCs, BM-MSCs, and i BM-MSCs transduced with control shRNAs (CTR) or with DNMT3B shRNAs (shDNMT3B) were delivered in HA/TCP powder (10⁶ cells in 10 mg) and transplanted underneath the dorsal skin of NOD/SCID mice for 8 weeks. Sections of transplants were analyzed by H&E and immunohistochemistry of DSP. Newly formed dentin (arrows) and odontoblasts (O.D.) were immunoreactive for DSP antibody in single-colony-derived DP-MSC transplants. HA HA/TCP, D dentin, CT connective tissue, B bone, A adipose tissue. Bar = 100 μm