Three-dimensional (3D) cultures of Ishikawa, HEC1A, and AN3CA cells. (a) Real-time RT-PCR for AF-6/afadin using cDNA from Ishikawa, HEC1A, and AN3CA cells, standardized with GAPDH. AF-6/afadin mRNA levels in 3D culture were significantly higher than that in monolayer (Mono) culture in the Ishikawa cells, remained unchanged from that in Mono culture in the HEC1A cells, and was significantly lower than that in Mono culture in the AN3CA cells. Significant difference were indicated as ** for P < 0.01. (b) AF-6/afadin immunohistochemistry of 3D culture showed that Ishikawa, HEC1A, and AN3CA cells were strongly positive, weakly positive and very weakly positive, respectively. A large portion of the cell clumps formed round gland-like structures in 3D cultures of Ishikawa cells, whereas in 3D cultures of HEC1A and AN3CA cells, irregular gland-like structures and in the form of disorganized colonies with no gland-like structures, respectively, were observed.