Fig. 1.

Thyroid cells proliferate and form polarized follicle-like structures in 3D Matrigel culture. (A) Follicular structure was confirmed by examining confocal z-sections from the top to the bottom throughout a representative fixed follicle-like structure that had been grown for 7 days in 3D Matrigel culture. (B) Quantitative analysis of follicular growth is depicted as the mean diameter of FRT follicle-like structures±s.d. after the indicated days of culture in Matrigel (n>20 structures). For each time point, the diameters were measured in the central section of brightfield images using a micrometer. (C) Cell proliferation during follicle morphogenesis was evaluated at the indicated days of culture in Matrigel after fixation and immunostaining with an antibody against Ki-67. The graph shows the quantitative analysis of cell proliferation depicted as the percentage (%) of follicle-like structures with 2–55 Ki67-positive cells±s.d. after the indicated days of culture in Matrigel (n>20 follicles). Representative fields illustrate the decreasing quantity of Ki67-positive nuclei in follicles from day 8 and rare Ki67-positive cells at day 10. (D) Apical–basal polarity establishment was observed at day 7 in FRT follicle-like structures that had been fixed and immunostained with antibodies against the tight junction protein ZO-1 (green), the adherens junction protein β-catenin (β-cat, red), the apical protein ezrin (green) and the cis-Golgi matrix protein GM130 (green). All nuclei were stained with DAPI (blue). Scale bars: 10 µm.