Figure 3: DJ-1 induces angiogenesis through FGFR-1 activation.

(a) DJ-1 induces FGFR-1 phosphorylation in iHUVECs. Cells were treated with DJ-1 (10 nM), FGF-2 (20 ng ml⁻¹) or VEGF (20 ng ml⁻¹) and phosphorylations of FGFR-1, VEGFR-2, Src, FAK and ERK1/2 were examined. (b) iHUVECs were pretreated with SU5402 (1 μM) for 30 min, and the cells were incubated with DJ-1 (10 nM), FGF-2 (20 ng ml⁻¹) or VEGF (20 ng ml⁻¹) for 6 h to analyse cell migration. NS, not significant and *P<0.05 versus DJ-1 or FGF-2 alone. (c,d) SU5402 (1 μM) abolishes iHUVECs tube formation stimulated by DJ-1. Photographs (X50) were taken at 9 h after treatment with DJ-1 (10 nM), FGF-2 (20 ng ml⁻¹) or vehicle control. The scale bar represents 400 μm. Tube area was also quantified as in d. **P<0.01 versus the vehicle or DJ-1 alone. (e) Shown are representative immunoblots of iHUVECs that were treated with DJ-1 (10 nM) for 5 min in the presence or absence of SU5402 (1 μM). DJ-1-stimulated phosphorylations of FGFR-1, Src, FAK and ERK1/2 were blocked by SU5402 treatment. (f) Knockdown of FGFR-1 or VEGFR-2 by siRNA was confirmed by immunoblot analysis. Cells were harvested at 72 h after transfection with siRNAs (100 nM) for non-targeting, FGFR-1-targeting or VEGFR-2-targeting. (g) The effects of FGFR-1 knockdown on DJ-1-induced iHUVEC migration. iHUVECs were transfected with siRNAs for non-targeting, FGFR-1 or VEGFR-2. At 72 h after transfection, cells were trypsinized and migration activity was investigated. DJ-1 (10 nM), FGF-2 (20 ng ml⁻¹) or VEGF (20 ng ml⁻¹) was treated for 6 h in the cell migration assay. NS, not significant and **P<0.01 versus non-targeting siRNA treatment. n=3 for all groups. All data are expressed as means±s.e.m.