Figure 3.

FIP5 is required for the establishment of the apical lumen during the early stages of epithelial cyst formation. (A–G) MDCK-shFIP5 cells were grown for 74 h in the presence (C, F, and G) or absence (A, B, D, and E) of 1 µg/ml of dox to pre-knockdown FIP5. Cells were then seeded in 3D cultures and grown for 24 h. Cells were fixed with 4% paraformaldehyde and stained with anti-gp135 (A–C, E, and G, green), anti-cingulin (D–G, red), or anti-FIP5 (B, red) antibodies. (H) The quantitation of 3D cyst polarization at the two and four cell stages from the experiment shown in A–F. Data shown are the means and standard deviations derived from three independent experiments. n is the number of cysts analyzed. (I) The quantitation of fully matured (9 d) epithelial cysts with a single lumen in cells incubated with or without 1 µg/ml of dox supplementation beginning 24 h after seeding in 3D cultures. Data shown are the means and standard deviations derived from three independent experiments (error bars). n is the number of cysts analyzed. Insets show the extent of FIP5 (green) knockdown in the presence of doxycycline. Bars: (A–G) 8 µm; (I) 20 µm.