Overexpression of nonmembrane-bound Gαi1 leads to spindle misorientation and defective cystogenesis. (A) Overexpression of Venus-Gαi1 induces multiple lumens but does not affect cell polarization during cystogenesis. Stable Tet-Off MDCK cells expressing Venus-Gαi1 were plated in matrigel in the absence of doxycycline for 4 d. Cysts were fixed and immunostained for aPKC, ZO-1, and β-catenin (red). Actin was stained with rhodamine phalloidin (red). 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. Bar, 20 µm. (B) Quantitation of cysts with single normal lumen from MDCK cells expressing Venus (control) or Venus-Gαi1 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; **, P < 0.05. (C) Scatter diagram of metaphase spindle angles in Venus-expressing control cysts or in Venus-Gαi1–expressing cysts grown in the absence (−Dox) or presence (+Dox) of 20 ng/ml doxycycline. Data were presented as in Fig. 3 C.