Ndel1 localizes at subdistal appendages of centrioles and suppresses unscheduled primary cilia assembly in proliferating RPE1 cells. (A) Ndel1 localization at the centrosome. RPE1 or HeLa cells were stained with the indicated antibodies. Each merged image is shown with cartoons; MC and DC indicate a mother centriole (possessing distal and subdistal appendages at the distal end) and a daughter centriole (without appendages), respectively. The entire cell images are shown in Fig. S1 B. (B–H) Proliferating RPE1 cells were transfected with each Ndel1- or NDE1-specific siRNA (target sequence 1 or 2) or control siRNA. For rescue experiments (F–H), we used RPE1 cells expressing Ndel1 isoform B (see Fig. S3 A) or NDE1 with C-terminal myc tag and silent mutations (to resist siRNA-mediated Ndel1 knockdown; Ndel1-Myc or NDE1-Myc) in a doxycycline (Dox)-dependent manner. 4 h after the transfection, the medium was changed to a new growing medium with (+) or without (−) 3 ng/ml Dox. The cells were collected at 48 (B–H) or 72 (B) h after transfection for the following analyses. The siRNA-treated cells were subjected to the immunoblotting (B and G), the immunostaining (B, C, E, F, and H) or TEM (D). GAPDH was used as a loading control (B and G). Asterisks indicate nonspecific bands caused by overproduction (G). Insets show magnified images of indicated centrosomes in lower micrographs (C and H). As control ciliated cells, we incubated RPE1 cells in the serum-free medium for 48 h (serum [−] in D and E). Exogenous Ndel1 or NDE1 was detected with anti-Myc antibody (F–H). Based on immunostaining patterns of anti–acetylated tubulin (ac-tub; C and H), we calculated the percentage of ciliated cells (B and F). Data represent mean ± SEM of three independent experiments (n > 100 each). *, P < 0.05; **, P < 0.01; and ***, P < 0.001, two-tailed unpaired Student’s t test. Bars: (A and E) 1 µm; (C and H, main) 5 μm; (C and H, insets) 1 µm; (D) 0.2 µm.