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. 2011 Oct 3;195(1):71–86. doi: 10.1083/jcb.201011112

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© 2011 Willenborg et al.

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Figure 8. — SNX18 is required for the establishment of the apical lumen at the early stages of epithelial cyst formation. (A and B) MDCK-shSNX18 cells were grown for 9 d in the presence (A-c and A-d) or absence (A-a and A-b) of 1 µg/ml of dox. Cells were then fixed with 4% paraformaldehyde and stained with anti-gp135 (A-a and A-c) or anti-cingulin (A-b and A-d) antibodies. B shows the quantitation of epithelial cysts with a single lumen. Data shown are the means and standard deviations derived from three independent experiments (error bars). n is the number of cysts analyzed. Insets show dox+ cyst expressing myc-SNX18. (C–E) MDCK-shSNX18 cells were grown for 74 h in the presence (C-c, C-d, and C-f) or absence (C-a, C-b, and C-e) of 1 µg/ml of dox to pre-knockdown SNX18. Cells were then seeded in 3D cultures and grown for 24 h. Cells were fixed with 4% paraformaldehyde and stained with anti-gp135, anti-cingulin, or anti-SNX18 antibodies. E shows the quantitation of 3D cyst polarization at the two and four cell stages of the experiment shown in C. Data shown are the means and standard deviations derived from three independent experiments. n is the number of cysts analyzed. (D) The quantitation of fully matured (9 d) epithelial cysts with a single lumen in cells incubated with or without 1 µg/ml of dox added after 24 h 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 SNX18 (green) knockdown in the presence of dox. Bars: (A) 8 µm; (B) 16 µm; (C) 3 µm; (D) 16 µm.