In vitro differentiation of C666-1 spheres. A, microscopic observation of C666-1 cells cultured as nonadherent undifferentiated spheres and adherent cells under differentiation conditions for 36 and 72 h. B, flow cytometry analysis of CD133+ (upper) and Oct-4-positive (lower) cell populations in C666-1 spheres and sphere-derived adherent progeny at 36 and 72 h. C, quantitative real time PCR analysis of miR-200a, CTNNB1, and ZEB2 expressions. mRNA levels in sphere were normalized as 1. PE, phycoerythrin.