Fig. 6: SHCBP1 suppresses the GAP activity of RACGAP to GTP-RAC1.

A RACGAP1 expression in 19 cases of bladder cancer and paired adjacent tissues (data from TCGA). ***p < 0.001. B RT-qPCR analysis of RACGAP1 expression in twenty cases of bladder cancer and normal tissues; **p < 0.01. C Correlation between SHCBP1 and RACGAP1 mRNA expression in bladder cancer tissues. D Kaplan–Meier analysis of the relationship between RACGAP1 expression and DSS in the GSE13507 and GSE32548 cohorts. E, F GSEA based on the TCGA dataset showed that the indicated gene sets, including Rho-GTPase signaling and ubiquitination processes, were enriched in the SHCBP1-high group. NES: normalized enrichment score; FDR: false discovery rate. G Immunoblotting assay to assess the impact of SHCBP1 inhibition on RACGAP1 protein expression in T24 cells treated with cycloheximide (40 nM) at the indicated time points. H Pull-down and immunoblotting assays of the GTPase activity of RAC1 and RhoA after knockdown of RACGAP1 in T24 cells. I Pull-down and immunoblotting assays of the GTPase activity of RAC1 after SHCBP1 depletion in T24 cells. J The RACGAP1-mediated hydrolysis activity toward small GTPases was tested in T24 cells transfected with certain concentrations of HA-SHCBP1 and Flag-RACGAP1 in an in vitro GAP assay. *p < 0.05, **p < 0.01 and ***p < 0.001. K Immunoblotting analysis indicating the alteration of active RAC1 following Flag-RACGAP1 and HA-SHCBP1 ectopic expression in T24 cells. L Pull-down assays for RAC1 activity were prepared in shNC-, shSHCBP1- or RACGAP1 siRNA-treated T24 cells in the presence or absence of EGF, and the expression of the indicated proteins was analyzed by immunoblotting. M The GTP hydrolysis activity toward RAC1 was measured in shNC-, shSHCBP1- or RACGAP1 siRNA-treated T24 cells in the presence or absence of EGF in an in vitro GAP assay. ***p < 0.001. N Immunoblotting analysis showing SHCBP1 expression in 5637 cells transfected with HA-SHCBP1. O Transwell assays showing the effect of HA-SHCBP1 ectopic expression and EHop-016 (10 μM) on EGF-induced migration of 5637 cells. *p < 0.05, **p < 0.01, ns: not significant. P A schematic diagram of SHCBP1 promoting EGF-stimulated cell migration and invasiveness by reducing the hydrolysis activity of RACGAP toward GTP-RAC1 is shown.