LGN depletion disrupts mitotic spindle orientation during cystogenesis. (A) Control, LGN-KD1-7, and LGN-KD2-6 MDCK cells were cultured in matrigel for 3–4 d. Cysts were fixed and immunostained for α-tubulin (red). DNA was stained with Hoechst 33342 (blue). Single confocal sections through the middle region of the cysts are shown. Arrows indicate mitotic cells. Bars, 20 µm. (B) Diagram depicting spindle angle measurement. Cysts grown for 3–4 d were fixed and stained with anti–α-tubulin antibody (green) and Hoechst 33342 (blue). Confocal images of metaphase cells in the middle region of the cysts were collected. A line was drawn to connect the two spindle poles (thick black lines). Another line was drawn from the centroid of the apical domain to the midpoint of the spindle axis (thin black lines), and the acute angle (red) between the two lines was analyzed. (C) Scatter diagram of metaphase spindle angles in control, LGN-KD1-7, and LGN-KD2-6 cysts. Results were from three independent experiments. Pink dots indicate mean values, green dots indicate individual data points, and error bars represent the SEM of the total number of spindles analyzed.