Invasiveness of Ishikawa cells is enhanced by AF-6/afadin KD through phosphorylation of ERK1/2 and Src. Invasiveness was evaluated using the Matrigel invasion assay. For this assay, siRNA for control or AF-6/afadin was transfected into the Ishikawa or HEC1A cells. (a-d) The number of invasive cells increased significantly in AF-6/afadin KD Ishikawa and HEC1A cells. Significant differences are indicated as * for P < 0.05. (e) Expression of GAPDH was used as the internal control. Using siRNA-negative control or siRNA against AF-6/afadin, AF-6/afadin was effectively knocked down at the protein level. AF-6/afadin KD induced phosphorylation of ERK1/2 and Src in the Ishikawa cells, whereas expression of phosphorylated ERK1/2 and Src was already strong and was not up-regulated by AF-6/afadin KD in HEC 1A cells.