Fig. 8.

p-Tyr42 RhoA binds to the promoter of Vim gene, leading to increased expression of vimentin. (A) Cells (4T1) were treated with hydrogen peroxide (100 μM) for 12 h and ChIP sequencing was performed. Common genes of Wnt-target and the promoter of which p-Tyr42 Rho binds to are listed. (B) Vim map and promoter region of Vim, the promoter of which p-Tyr42 Rho was associated with was shown. (C) 4T1 cells were stimulated with Wnt3A (30 ng/ml) for 2 h and ChIP PCR was performed with primers covering the promoter region. (D) HEK293T cells were stimulated with Wnt3A (30 ng/ml) for 2 h and maker proteins for EMT were assessed by western blotting. (E) HEK293T cells were transfected with si-RhoA for 72 h, then transfected with RhoA WT and Y42F. Expressions of vimentin and RhoA were detected by western blotting of the cell lysates. (F) HEK293T cells were transfected with RhoA WT, Y42E and Y42F DNA (4 μg) along with Vim promoter-Luc DNA for 24 h, then cells were stimulated by Wnt3A (30 ng/ml) for 2 h and luciferase activity was measured. (G) HEK293T cells were transfected with si-control and si-β-catenin (10 nM) for 72 h, then cells were stimulated by Wnt3A (30 ng/ml) for 2 h and luciferase activity was subsequently measured. (H) Migration of cells transfected with RhoA WT, Y42E and Y42F were measured. (I) Survival probability of gastric cancer patients harboring low and high VIM mRNA levels was obtained from Kaplan-Meier Plotter site.