A CCD18-co, SW480, and HCT116 cells with PAK4 knockdown were established. PAK4 expression was determined by western blot. B MTT assays demonstrate that PAK4 knockdown reduces cell proliferation in CCD18-co, SW480, and HCT116 cells. C Anchorage-independent growth in different PAK4 knockdown cells. D Number of colonies determined by Image J-Plus (Scale bar: 200 μm). Data represents statistical analysis of the colony ratio. E The proliferation ability in cells transfected with different S291 mutations of PAK4 was determined by MTT assay. F Anchorage-independent growth in cells transfected with different PAK4 S291 mutations (Scale bar: 200 μm). Data represent statistical analysis of the colony ratio in right panel. G β-catenin and MEK/ERK signals determined by western blot in cells transfected with different PAK4 S291 mutations. Densitometric analysis represents the ratio of phosphorylated protein/total protein. Data were presented as mean values ± SD from triplicate experiments. Statistical differences were evaluated using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.