Fig 2: CCDC68 and CCDC120 competitively interact with CEP170.(a) Immunostaining of CCDC68 (green) and ?-tubulin (red) in control- or ODF2-siRNA-transfected U2OS cells. Quantification of CCDC68 at the SDAs are shown at right. (b) Immunoblots show depletion of CCDC68 and CEP170 by siRNA in U2OS cells. (c) Immunostaining of CEP170 (green) and ?-tubulin (red) in control- or CCDC68-siRNA-transfected U2OS cells. Arrow, centrosome localization. Quantification of CEP170 at centrosomes are shown at right. (d) Schematic of CCDC68. FL, full-length; coiled-coil domains, blue; +, positive; -, negative. (e) Lysates of HEK293T cells co-overexpressing GFP-CEP170 and Flag-CCDC68 truncates were subjected to immunoprecipitation (IP) and immunoblotted (IB) with the indicated antibodies. Asterisks, IgG bands. (f) Immunostaining of U2OS cells overexpressing Flag-CCDC68 (101–305 aa) with anti-Flag (green) and anti-CEP170 (red) antibodies. DNA, 4,6-diamidino-2-phenylindole (DAPI; blue). Centrosomes are magnified. Quantification of CEP170 at centrosomes is shown below. (g) Lysates of HEK293T cells co-overexpressing GFP-CEP170 and Flag-CCDC68 truncates were subjected to IP and IB with anti-GFP and anti-Flag antibodies. Asterisk, IgG. (h) Lysates of HEK293T cells overexpressing Flag-CCDC120 or Flag-CCDC68 were subjected to pull-down assays with MBP-CEP170-C2 (1,015–1,460 aa). Quantification of the Flag-CCDC68 and Flag-CCDC120 band intensity (normalized to MBP-CEP170-C2) in three independent experiments are shown on the graph. (i) Quantification of the fluorescence intensity of CEP170 at centrosomes in the indicated siRNA-transfected U2OS cells. (j) Immunoblots show depletion of CCDC68 and CCDC120 by siRNA in U2OS cells. (k) Immunostaining of CCDC120 (green) and ?-tubulin (red) in control- or CCDC68-siRNA-transfected U2OS cells. Arrow, centrosome localization. Quantification of CCDC120 at centrosomes are shown at right. (l) Immunostaining of CCDC68 (green) and ?-tubulin (red) in control- or CCDC120-siRNA-transfected U2OS cells. Arrow, centrosome localization. Quantification of CCDC68 at centrosomes are shown at right. For a,c,f,i,k and l scale bars, 1 µm. Data in a,c,f,i,k and l are the mean±s.e.m. n>100 cells from three individual experiments, Statistical significance was determined by a two-sided Student's t-test. *P<0.05, **P<0.01; NS, not significant. Unprocessed original scans of immunoblots are shown in Supplementary Fig. 7.

Fig 3: CCDC68 interacts with CEP170 and is localized at the centrosomes.(a) Immunoprecipitation (IP) of CCDC68 with CEP170 in lysates of HeLa cells. (b) Immunofluorescence of CCDC68 (green) and Centrin-3 (red) in U2OS cells. Scale bar, 500 nm. (c–f) 3D-SIM images of Flag-CCDC68-overexpressing U2OS cells double-immunostained with antibodies against Flag (green) and hNinein (c; red), CEP170 (d, red), CCDC120 (e, red) or ODF2 (f, red). Scale bars, 500 nm. The intensity plots of the rings are, respectively, shown at right. (g) Average diameter of the ring-like structure formed by the listed proteins. The low-high bars (horizontal) show the range of the diameter and the vertical lines indicate the mean. From the bottom to the top: n=10; 36; and 28. (h) Immuno-EM images. U2OS cells were labelled with anti-CCDC68 antibody followed by nanogold-coupled secondary antibody. Schematics of immuno-EM images are shown. Scale bar, 200 nm. Supplementary Table 1 lists the CEP170-interacting proteins.

Fig 4: Schematic model of the roles of CCDC120 and CCDC68 in hierarchical SDA assembly in interphase cells.(a) Schematic model of SDAs. SDA components ODF2, CCDC68, CCDC120, hNinein and CEP170 formed a ring-like structure at SDAs of the mother centrioles in interphase cells. (b) Schematic model of SDA assembly. ODF2 acts upstream to initiate SDA assembly2021. TCHP mediates the interaction between ODF2 and hNinein23. CEP170 can be recruited by hNinein19. CCDC120 is recruited to SDAs by ODF2 and recruits both CEP170 and hNinein to SDAs through two distinct domains. CCDC68 competes with CCDC120 in recruiting CEP170. (c) Schematic model of the interactions of CCDC68, CEP170, CCDC120 and hNinein, and their binding domains.