Figure 4: DJ-1 stimulates osteoblast differentiation and mineralization of hMSCs.

(a) hMSCs were treated with DJ-1 (0.1–100 nM) in osteogenic differentiation condition for 9 days. Osteogenic differentiation was confirmed by alizarin red S staining. (b) Osteogenic marker expression during osteogenesis was confirmed by quantitative reverse transcription-PCR. *P<0.05 and **P<0.01 versus the vehicle. (c) DJ-1 (10 nM) induced phosphorylations of FGFR-1 and ERK1/2 in hMSCs. (d) SU5402 (1 μM) treatment abolished the phosphorylation of FGFR-1 induced by DJ-1. (e) Effect of SU5402 on DJ-1-stimulated osteogenesis was confirmed by alizarin red S staining at 7 days after osteogenesis induction. (f) Quantification of the amount of alizarin red S staining. **P<0.01 versus the vehicle or DJ-1 alone. (g) hMSCs were transfected with non-targeting or two different DJ-1 shRNAs and subjected to immunoblot analysis. (h) Knockdown of DJ-1 by shRNA suppressed osteoblast mineralization, an effect which was confirmed by alizarin red S staining at 12 days after osteogenesis induction. GM indicates growth medium and osteogenesis induction medium (OIM) indicates osteogenesis induction medium. (i) Exogenous DJ-1 rescues osteoblast differentiation in DJ-1 knockdown cells and it was examined by alizarin red S staining at 9 days after osteogenesis induction. (j) Quantification of the amount of alizarin red S staining. **P<0.01 versus the vehicle of non-targeting shRNA treatment. n=3 for all groups. All data are presented as means±s.e.m.