DAs are released from interphase centrosomes after ectopic Nek2 overexpression. (A) Immunofluorescence of the analyzed appendages in RPE1 cells carrying mNeonGreen-Nek2A (WT) and mNeonGreen-Nek2A-K37R (KD mutant) under control of the DOX-inducible promoter. γ-tubulin labels centrosomes, and DAPI stained the DNA. Insets inside each panel show a magnification of the centrosomal area and enlargements for the indicated staining are shown on top of each panel. Scale bars, 20 µm (panel) and 2 µm (inset). (B) Boxplots show the quantification of DAs at the centrosome in control (− DOX) and Nek2A-overexpressing (+ DOX) samples of A. The control Nek2 WT−DOX sample was used for normalization of three independent experiments (n = 150 cells per condition). For LRRC45 analysis, C-Nap1 was depleted using CEP250 siRNA before DOX treatment and siC-Nap1 (WT−DOX) samples were used for normalization. (C) RPE1 WT and mNeonGreen-Nek2A–expressing cells were treated with the proteasome inhibitor MG132 (+ MG) or solvent only (− MG) during the last 4 h of DOX treatment and analyzed as described in B. n = 150 cells per condition in three independent experiments. Boxes show interquartile range, lines inside the box represent the median, and whiskers show minimum and maximum values excluding outliers. 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.