Nek2-induced centrosomal appendage release is independent of ODF2. (A and B) Images of RPE1 WT and ODF2 KO cells stained with centriolin, ODF2 (A) and Cep128 (B) antibodies. Scale bar, 5 µm. (C) Images show RPE1 WT and ODF2 KO interphase cells stained for the indicated DAs. Box/dot plots show the normalized quantification of DA signal at the centrosome in RPE1 WT and ODF2 KO cells. Dots represent individual cells, boxes show interquartile range, lines inside the box represent the median, and whiskers show minimum and maximum values excluding outliers. Combined data from three independent experiments are shown (n = 150 cells per condition). Scale bar, 5 µm. (D) Representative images of mitotic RPE1 WT and ODF2 KO cells stained for Cep164. Scale bar, 20 µm. (E) Effect of Nek2A overexpression upon Cep164 in the absence of ODF2. Box/dot plots show quantification of Cep164 intensity in RPE1 mNeonGreen-Nek2A cells treated with control or ODF2 siRNA in the presence (+ DOX) or absence (− DOX) of DOX. Combined data from three independent experiments are shown (n = 150 cells per condition). (F) Images of mitotic RPE1 ODF2 KO cells treated with control (Ctrl) or Nek2 siRNA and stained with Cep164 antibodies. The graph shows the percentage of mitotic cells in which Cep164 associates at one centrosome (arrow in ODF2 KO siNek2 sample, asymmetric Cep164). Bar graphs represent mean ± SD. Scale bar, 20 µm. n = 128 (siCtrl) and n = 108 (siNek2) in two independent experiments. γ-tubulin (γ-tub) and DAPI serve as markers for centrosomes and nuclei, respectively. A.U., arbitrary units; n.s., not significant. Significance probability values are: n.s., P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.