Figure 4

Involvement of HIF-1α in WISP-1-induced VEGF-A expression and angiogenesis (a and b) MG-63 cells were pretreated with an HIF-1 inhibitor (HIF i; 10 μM) for 30 min or HIF-1α siRNA for 24 h, then treated with WISP-1 (30 ng/ml) for 24 h. mRNA and VEGF-A protein expression was detected by RT-qPCR and ELISA. Untreated cells were used as the control. (c) MG-63 cells were pretreated with an HIF i (10 μM) for 30 min and then treated with WISP-1 (30 ng/ml) for 24 h. Culture medium was collected as CM and then applied to EPCs for 24 h. EPC capillary-like structure formation was examined by tube formation (bar=100 μm). CM collected from untreated cells was used as the control. (d) MG-63 cells were incubated with WISP-1 (30 ng/ml) for the indicated times and HIF-1α expression was detected by western blot (MW: molecular weight). (e) MG-63 cells were treated with WISP-1 (30 ng/ml) for 8 h, then incubated for 0–60 min with CHX 5 μM. HIF-1α protein expression was detected by western blot (MW: molecular weight) and the quantification results are shown in (f). (g) MG-63 cells were pretreated with HIF i or SP600125 for 30 min, then treated with WISP-1 (30 ng/ml) for 24 h. HIF-1α protein expression was detected by western blot (MW, molecular weight). Untreated cells were used as the control. (h) MG-63 cells were pretreated with HIF i and SP600125 for 30 min or pre-transfected with FAK and JNK siRNA before exposure to WISP-1. HRE-luciferase activity was measured, and the results were normalized to β-galactosidase activity. Untreated cells were used as the control. Each experiment was performed in triplicate (N=3). Results are expressed as the mean±S.E.M. *P<0.05 compared with control; ^#P<0.05 compared with the WISP-1-treated group