Figure 1.

NEDD1 facilitates cartwheel assembly initiation and stabilizes the cartwheel structure. (A) U2OS cells treated with siRNA against NEDD1, CDK5RAP2, or γ-tubulin were coimmunostained with antibodies against NEDD1, CDK5RAP2, or γ-tubulin and SAS-6. C5R2, CDK5RAP2. DNA was stained with DAPI. Scale bars, 5 µm (large images) or 0.5 µm (inset images). (B) Comparisons of relative fluorescence intensity of SAS-6 in A. Three independent replicates of >30 cells per replicate were quantified. (C) U2OS cells treated with siRNA against control or NEDD1 were coimmunostained with antibodies against NEDD1 and STIL, PLK4, Cep152, or Cep192. DNA was stained with DAPI. Scale bars, 5 µm (large images) or 0.5 µm (inset images). (D) Comparisons of the relative fluorescence intensity of related proteins in C. Three independent replicates of >30 cells per replicate were quantified. (E) HEK293T cells were transfected with GFP-STIL and treated with control, NEDD1, or γ-tubulin siRNA. Total cell extracts were immunoprecipitated with GFP-Trap beads and probed with antibodies against SAS-6, GFP, NEDD1, γ-tubulin, and α-tubulin. (F) Immunofluorescence of NEDD1 and γ-tubulin, SAS-6, STIL, or Myc in U2OS cells transfected with Myc-PLK4. The stained proteins’ centrosomal features are shown in the cartoons below each image. DNA was stained with DAPI. Scale bars, 5 µm (large images) or 0.5 µm (inset images). (G) Quantification of outer toroid/ring diameters (with means) of the indicated cartwheel or PCM proteins in U2OS cells at interphase. Three independent replicates of >15 cells per replicate were quantified. (H) Organizational features of the interphase PCM components. Error bars in B, D, and G indicate mean ± SD. ***, P < 0.001; ns, no significant difference (two-tailed t test). NC, negative control; Tub, tubulin.