Figure 4

Cyr61-promoted EMT is mediated by a Raf-1/MEK/ERK signaling cascade. (A) MG63 cells were pretreated with Gw5074, PD98059 or U0126 for 30 min followed by stimulation with Cyr61 (10 ng/mL) for 24 h. Cell migration was measured after 24 h using the Transwell assay. (B and C) MG63 cells were treated as in (A). Total mRNA and proteins were extracted from the MG63 cells, and the expression levels of TWIST-1, N-cadherin, and E-cadherin were detected using qRT-PCR and western blotting. (D) MG63 cells were incubated with Cyr61 (10 ng/mL) for the indicated times, and the phosphorylation of Raf-1, MEK, and ERK was determined through western blotting. (E) MG63 cells were transfected with Raf-1 shRNA, DN-MEK, DN-ERK, or a control vector for 24 h, followed by treatment with Cyr61 (10 ng/mL) for 24 h, and cell migration was analyzed using the Transwell assay. (F and G) MG63 cells were treated as in (E). Total mRNA and proteins were extracted from the MG63 cells, and the expression levels of TWIST-1, N-cadherin, and E-cadherin were detected using qRT-PCR and western blotting. (H) MG63 cells were pretreated with anti-integrin αvβ3 or αvβ5 mAb (3 μg/mL) for 30 min, followed by stimulation with Cyr61 (10 ng/mL) for 10 min, and Raf-1 phosphorylation was determined with western blotting. (I) MG63 cells were pretreated with Raf-1 inhibitor (Gw5074) (5 μM) for 30 min, followed by stimulation with Cyr61 (10 ng/mL) for 30 min, and MEK phosphorylation was determined with western blotting. The results are expressed as the mean ± SEM of triplicate samples. *P <0.05 compared with the control group, and ^#P <0.05 compared with Cyr61 treatment.