Overexpression of the C terminus of LGN or the LGN-binding domain of NuMA leads to spindle misorientation and defective cystogenesis. (A and B) Overexpression of the GoLoco-repeat C terminus of LGN (A) or the LGN-binding domain of NuMA (B) induces multiple lumens but does not affect cell polarization. Stable Tet-Off MDCK cells expressing Venus-LGN476-CT (A) or Venus-NuMA1818-1921 (B) were plated in matrigel in the absence of doxycycline for 4 d. Cysts were fixed and immunostained for aPKC, ZO-1, β-catenin, NuMA, or LGN (red). Actin was stained with rhodamine phalloidin (red, top). DNA was stained with Hoechst 33342 (blue). Single confocal sections through the middle region of the cysts are shown. Merged images are shown on the right. Bars, 20 µm. (C) Quantitation of cysts with single normal lumen from MDCK cells expressing Venus (control), Venus-LGN476-CT, or Venus-NuMA1818-1921 in the absence (−Dox) or presence (+Dox) of 20 ng/ml doxycycline. Values are mean ± SD from three independent experiments; n > 100 cysts/experiment. *, P < 0.01. (D) Scatter diagram of metaphase spindle angles in cysts expressing Venus (control), Venus-LGN476-CT, or Venus-NuMA1818-1921 in the absence (−Dox) or presence (+Dox) of 20 ng/ml doxycycline. Data were presented as in Fig. 3 C.