Knockdown of LGN in MDCK cells results in defective cystogenesis. (A) Western blot analysis of representative cell lines stably transduced by control or two different lentiviruses expressing shRNAs against LGN (LGN-KD1-7 and LGN-KD2-6). Total cell lysates were blotted for LGN and α-tubulin. The asterisk indicates nonspecific bands. (B) The cortical staining of LGN in mitotic cells is significantly reduced in LGN knockdown cells. Control, LGN-KD1-7, and LGN-KD2-6 MDCK cells were fixed and immunostained for LGN (red). DNA was stained with Hoechst 33342 (blue). The merged images are also shown (right). Note that these lentivirus-transduced cell lines express GFP. Arrows point to mitotic cells. (C) Knockdown of LGN induces multiple lumens but does not affect cell polarization. Control (left panels) or LGN KD1-7 (right panels) cells were plated in matrigel for 3–4 d. Cysts were fixed and immunostained to detect aPKC, Pals1, ZO-1, β-catenin, and Dlg1 (red). Actin was stained with rhodamine phalloidin (red, top). Single confocal sections through the middle region of the cysts are shown. Bars, 20 µm. (D) Quantitation of cysts with single normal lumen in control, LGN KD1-7, or LGN KD2-6 cells. Values are mean ± SD from three independent experiments; n > 100 cysts/experiment. *, P < 0.01.