Fig 2: WT Mig-6, but not the 4A mutant, inhibits Cdc42 activation(A) H1299 cells stably expressing Mig-6-FL or FL-4A mutant were subjected to western blotting, as shown. (B) H1299 cells stably expressing CRIB or CRIB-4A were subjected to western blotting. (C) H1299 cells stably expressing shRNA specific for Mig-6 (shMig-6) or a scrambled shRNA sequence (shC) were subjected to western blotting. (D) Ectopic expression of Cdc42 reverses Mig-6 inhibitory effect on EGF-induced cell migration. H1299 cells stably expressing Myc-Mig-6 were infected with lentivirus expressing HA-Cdc42 or vector control. H1299 cells stably expressing mutant Myc-Mig-6(4A) was used in parallel. Cell lysates were subjected to western blotting, as indicated. (E) Stable cells were subjected to transwell assays in the presence of EGF for 8 hours. Representative pictures are shown. Scale bars = 100 µm. (F) Results from three independent migration assays were quantitated and presented as means and SE. ***P < 0.001.

Fig 3: CIP4 directly interacts with activated Cdc42 to accelerate invadopodia function(A) Co-localization of CIP4 (green) and Cdc42 (red) in Lovo and HT29 cells was observed by immunofluorescence. Magnification: 180×. The Pearson’s correlation and overlap coefficient were shown in bar graph format. The error bars represent the mean ± SD (n = 5). (B) GST pull down experiments on CIP4 protein with mutants of activated Cdc42. GST and HIS were analyzed by western blot. (C) The localization of CIP4 and invadopdia was observed in Lovo and HT29 cells by confocal laser scanning. The white arrowhead indicates the co-localization of CIP4 and invadopodia. Magnification: 180×. (D) The localization of FLAG-tagged activated Cdc42 and invadopdia was observed in CIP4 overexpression or low-expression cells by confocal laser scanning. The white arrowhead indicates the co-localization of FLAG-tagged activated Cdc42 and invadopodia. Magnification: 180×.

Fig 4: Inhibition of CDC42 prevents NDRG1 loss induced CRC cell filopodial protrusion formation through suppression of PAK1/Cofilin signaling. A) Immunoblotting analysis of the expression level of the total and phosphorylation form of PAK1 and Cofilin in indicated cell lines. B) Knockdown of CDC42 in HCT116 (left) and RKO (right) cells confirmed with immunoblotting analysis. Pool, combined siCDC42 sequences. C) Expression level of the total and phosphorylation form of PAK1 and Cofilin in indicated cell lines. D) Confocal images were taken to show immunofluorescence staining of MYO10 (green) and rhodamine-phalloidin (red) accompanied by the cell nucleus (blue) in colorectal cancer cells. Quantification of the MYO10-associated filopodial protrusions density and length is represented as mean ± S.D.; results are representative of 3-5 images from different visual fields, n>50 cells. *P value <0.05, **P value <0.01, ***P < 0.001, relative to the sh-Con/si-Con groups. #P value <0.05, ##P value <0.01, ###P < 0.001, relative to the sh-NDRG1/si-Con groups.

Fig 5: CDC42GTP is frequently upregulated in CRC tissues and correlated with NDRG1 expression and clinicopathological parameters. A) IHC staining of NDRG1 and active CDC42 expression in tumor and adjacent tissues in microarray. Magnification on the right with a scale bar of 100 µm. B) Heatmap illustrating different clinicopathological parameters between CDC42GTP-high and -low-expression tumors of the 86 cases. Statistical significance was analyzed by the ?2 test. P values are as indicated.