Effects of PHD inhibitors on rosiglitazone-induced anti-osteoblast differentiation.

<p>Primary bone marrow cells were treated with either DMOG (1 mM) or EDHB (20 μM) in the presence or absence of rosiglitazone (10 μM) for 12 days (media were changed every 48 h). Extents of osteoblast differentiation were monitored by Alizarin red staining (A), and the mRNA levels of the PHD 1–3 and osteomarker marker genes were determined by RT-PCR (B). The protein levels of Runx2 and levels of Runx-2 ubiquitination were determined by western blotting (C) and by immunoprecipitation using polyubiquitination antibody (D). The effect of MG-132 (5 μM; an inhibitor of proteasomal degradation) on Runx-2 expression was examined by western blotting (E). Experiments were conducted three times and representative results are shown. Densitometric analysis was conducted using UN-SCAN-IT gel ver. 5.1 software (Silk Scientific) and is expressed as means ± SEMs of three experiments. *<i>P</i><0.05 vs control (C), #<i>P</i><0.05 vs differentiated (D), ✝<i>P</i><0.05 vs DR. C, control (no differentiation); D, differentiated in DAG medium; DR, DAG in the presence of rosiglitazone; DM, DMOG; ED, EDHB; MG, MG-132.