Figure 5

Cyr61-promoted EMT is mediated by Elk-1 transcription factor. (A) MG63 cells were incubated with Cyr61 (10 ng/mL) for the indicated times, the nuclear and cytosol extract were prepared, and the phosphorylation of ERK and Elk-1 was determined with western blotting. (B) MG63 cells were transfected with Elk-1 shRNA, or a control vector for 24 h, followed by treatment with Cyr61 for 24 h, and cell migration was analyzed using the Transwell assay. (C) MG63 cells were treated as in (B). Total proteins were extracted from the MG63 cells, and the expression levels of TWIST-1, N-cadherin, and E-cadherin were detected with western blotting. (D) MG63 cells were pretreated with Gw5074 for 30 min, followed by stimulation with Cyr61 (10 ng/mL) for 60 min, and the nuclear and cytosol extract were prepared, and ERK phosphorylation was determined with western blotting. (E) MG63 cells were treated as in (D). Cells were stained with an anti-ERK antibody and analyzed using fluorescence microscopy. Nuclei were counterstained with DAPI. Representative confocal microscopy images are shown. (F) MG63 cells were pretreated with Gw5074, PD98059, or U0126 for 30 min, followed by stimulation with Cyr61 (10 ng/mL) for 60 min, the nuclear extract was prepared, and Elk-1 phosphorylation was determined with western blotting. (G) MG63 cells were treated as in (F). Chromatin immunoprecipitation was performed using anti-Elk-1. Immunoprecipitated chromatin (1%) was assayed to verify equal loading (input). 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.