Morphology change induced by AF-6/afadin KD in the Ishikawa cells in 3D culture. The paraffin-embedded samples were cut into 3.5-μm sections. Immunohistochemistry was performed to confirm the efficacy of AF-6/afadin KD (data not shown) and (a) we also examined AF-6/afadin KD efficacy by western blotting. (b, c) The morphological differences in 3D cultures were assessed by hematoxylin-eosin staining. The KD control Ishikawa cells mainly formed the glands like structures. Panel (c) is the magnification of the box in (b). (d, e) AF-6/afadin KD Ishikawa cells mainly formed disorganized colonies. Panel (e) is magnification of the box in (d). (f) The number of gland-like structures per 100 cell clumps was counted three times. AF-6/afadin KD significantly reduced the number of gland-like structures. Significant differences were indicated as ** for P < 0.01.