Figure 4.

ERK1/2 (extracellular signal-regulated kinases 1/2) and PI3K (phosphatidylinositol 3-kinase)/AKT signaling pathways act downstream of Sca-1 (stem cell antigen-1)+ cell migration mediated by Dkk3 (dickkopf-3) binding to CXCR7. A and B, Western blot analysis of phosphorylated and total ERK1/2 and AKT proteins in Sca-1+ cells stimulated with Dkk3 at the indicated time points. ERK1/2 and AKT phosphorylation increases in response to Dkk3. C and D, Western blot analysis of Dkk3-induced ERK1/2 and AKT activation in Sca-1+ cells after CXCR7 knockdown by siRNA transfection. Downregulation of CXCR7 abolishes Dkk3-triggered phosphorylation of ERK1/2 and AKT. E and F, Western blot analysis of ERK1/2 and AKT activation in Sca-1+ cells stimulated with Dkk3 for 5 min and pretreated with PD98059 and LY294002, respectively. ERK1/2 and AKT activation is supressed on treatment with the inhibitors. G–I, Quantification of Dkk3-driven Sca-1+ cell migration on treatment with PD98059 (10 µmol/L; n=5), LY294002 (10 µmol/L; n=3), and AKT inhibitor (2.5 µmol/L; n=4), respectively. Dkk3-driven cell migration is abrogated in response to all inhibitors. Western blot images are representative of 3 independent experiments. The data are expressed as the mean±SEM of 3 or 5 independent experiments. *P<0.05, **P<0.01, ***P<0.001, compared with control group (0 ng/mL; 2-way ANOVA followed by Bonferroni post hoc test).