Forced apical targeting of endogenous LGN reverses spindle orientation and leads to defective cystogenesis. (A) Stable inducible MDCK cells expressing Crb3-Venus-Gαi1wt were cultured in matrigel in the absence of doxycycline for 4 d. Cysts were fixed and immunostained for LGN, NuMA, and tubulin (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. Enlarged images of highlighted mitotic cells (square boxes) are shown on the right. Bars, 20 µm. (B) Quantitation of cysts with single normal lumen from MDCK cells expressing Crb3-Venus, Crb3-Venus-Gαi1N149I, or Crb3-Venus-Gαi1wt 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 < 0.001; **, P < 0.01. (C) Apical targeting of the GoLoco-insensitive Gαi1 mutant (Gαi1N149I) does not affect cystogenesis, localization of LGN and NuMA, or mitotic spindle orientation. Stable MDCK cells expressing Crb3-Venus-Gαi1N149I were plated in matrigel in the absence of doxycycline for 4 d and analyzed as in A. (D) Scatter diagram of metaphase spindle angles in cysts expressing Crb3-Venus, Crb3-Venus-Gαi1wt, or Crb3-Venus-Gαi1N149I. Data were presented as in Fig. 3 C.