aPKC activity prevents apical localization of LGN during cystogenesis. (A) Inhibition of aPKC activity leads to apical diffusion of LGN. MDCK II cells were plated in matrigel for 3 d to allow cystogenesis. Cysts were treated with aPKC-PS (40 µM final concentration) for 24 h or left untreated. Treated (+aPKC-PS) or untreated (−aPKC-PS) cysts were then fixed and immunostained for LGN (green). 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. Bars, 20 µm. (B) Overexpression of active PKC-ζ inhibits cortical localization of LGN. MDCK cells were transfected with pK-myr-Venus, pK-myr-aPKCζwt-Venus, or pK-myr-aPKCζK281W-Venus and cultured for 24 h. Cells were fixed and immunostained with anti-LGN antibody (left). DNA was stained with Hoechst 33342 (right). Bar, 20 µm. (C) Quantitation of the relative fluorescence intensity of cortical LGN in cells expressing myr-Venus, myr-aPKCζwt-Venus, or myr-aPKCζK281W-Venus. Fluorescence intensities at the cortex and 10 pixels away at the cytosol were refered to as F(cortex) and F(cytosol), respectively. The ratio of F(cortex)/F(cytosol) was collected for each group of cells and analyzed. Values are mean ± SD from three independent experiments; n > 10 cells/experiment. *, P < 0.0001.