Expression of osteoblast inhibitors in coculture. Human BM PreOBs were cocultured in the presence or absence of a transwell insert with OPM2. (A) After 24 hours, mRNA was extracted and the expression of noggin; gremlin sFRP-1, sFRP-2, sFRP-3, and sFRP-4; and DKK1 was evaluated by RT-PCR. (B) sFRP-2, sFRP-3, and DKK1 protein expression was evaluated by Western blot analysis after 48 hours. Figures are representative of 3 independent experiments. Graphs represent the mean optic density (OD ratio) of DKK1 normalized to actin of a representative experiment. The following positive controls (Con +) have been used: noggin, human amniotic cells; Gremlin, osteoblast-like cell line MG-63 stimulated with rhBMP-2 (100 ng/mL), sFRP-1, sFRP-2, sFRP-3, and sFRP-4; fetal and adult human cardiomiocytes, DKK1; and HOBIT.