Figure 5.

E620K mutation of ARHGEF12 promotes OM in GC. (A) WB of ARHGEF12 knockout (KO) and ARHGEF12 mutants. A control sgRNA (sgCtrl) and one ARHGEF12 sgRNA (sgARHGEF12) were used. Empty vector (EV), wild-type (WT) ARHGEF12 or ARHGEF12 mutants were re-expressed in ARHGEF12 KO AGS cells. ARH12, ARHGEF12. (B-D) Effects of ARHGEF12 mutations on cell migration (B), invasion (C) and colony formation (D). (E-G) Effects of ARHGEF12 E620K mutation on cell migration (E), invasion (F) and colony formation (G) in MKN45 cells. (H) Quantification of filopodia numbers affected by ARHGEF12 E620K mutation in AGS and MKN45 cells. Data shown are from 30 cells counted per condition. (I) Immunoblotting for the active RHOA by Rhotekin pulldown assay. Cells were serum starved for 24 h and stimulated with 10% FBS for 10 min. Total cell lysates were subjected to GST-Rhotekin Rho-binding domain (RBD) precipitation and then the activities of RhoA were examined. (J) Kaplan-Meier survival of animals with indicated AGS tumors (n = 10). (K) Representative bright-field (upper panel) and hematoxylin and eosin (H&E) staining (lower panel) images of the animal lungs. Scale bars, upper, 300 µm, lower, 100 µm. (L) The numbers of nodules on the lung surfaces in (K) quantified by necropsy. (M) Representative H&E staining images of the animal livers with non-metastasis and metastasis tumors. Scale bars, 100 µm. (N) Representative H&E staining images of the animal ovaries with non-metastasis and metastasis tumors. Scale bars, 20 µm. (O) Detailed summary of the tumorigenicity and peritoneal metastasis of animals. Data are representative of three independent experiments with similar results. Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by two-tailed Student's t-test or log-rank test.