Tumor invasion assay of colon cancer cell lines with a Matrigel-coated invasion chamber. (A) Expression of ARK5 in cell extracts from DLD-1, D/ARK, and SW480 cells was assessed. (B) One microgram of double-stranded RNA with (+) or without (−) ARK5i was exposed to the D/ARK (DA) or the SW480 (SW) cell lines, and Western blotting was performed. (C) Phosphorylation of Akt in cell lines exposed or not exposed to 100 ng of IGF-1/ml for 2 h was also assessed by Western blotting. (D and E) The invasion activity of SW480 (D), DLD-1 (E) (open bars), and D/ARK (E) (closed bars) cell lines exposed to 20 μM LY294002, transiently transfected with 1 μg of DN-Akt1, DN-ARK5, control RNAi (CNTi), or ARK5i, in the presence or absence (Cont) of 100 ng of IGF-1/ml for 48 h was assessed. The numbers of invading cells are the means of data from three experiments, and bars represent the standard errors.