Asef-mediated, Asef2-mediated and APC-mediated upregulation of MMP9 is essential for invasion of colorectal tumour cells. (A) Cells were transfected with vectors expressing the indicated shRNAs and tested in invasion assays. Results obtained are expressed as the means±s.e.m. of at least four independent experiments. **P<0.01; ***P<0.001. (B) Exogeneous expression of MMP9 restores the invasive activity of SW480 cells in which Asef2 and APC had been knocked down. SW480 cells were transfected with siRNAs for Asef2 or APC together with MMP9, and subjected to immunoblotting with the indicated antibodies, gelatin zymography for MMP9 and invasion assays. Transfection efficiency was routinely more than 70%. Results are expressed as the means±s.e.m. of four independent experiments. *P<0.05. APC, adenomatous polyposis coli; MMP9, matrix metalloproteinase 9; shRNA, short hairpin RNA; siRNA, small interfering RNA; zymo, gelatin zymography.