Figure S3.

Both NEDD1 and γ-tubulin are necessary for centrosome duplication. (A) U2OS cells treated with negative control or NEDD1 siRNA were coimmunostained with antibodies against NEDD1 and Centrin1. DNA was stained with DAPI. Scale bars, 5 µm (large images) or 0.5 µm (inset images). (B) Percentages of the cells with different numbers of centrioles and comparisons of those cells with fewer than four centrioles in each treatment in A. Three independent replicates of >100 cells per replicate were quantified. (C) U2OS cells transfected with the corresponding siRNA and treated with 16 mM HU to allow centriole amplification were then stained with antibodies against NEDD1 and Centrin1. DNA was stained with DAPI. Scale bars, 0.5 µm. (D) Percentages of the cells with different numbers of centrioles and comparisons of those cells with >4 centrioles in each treatment in (C). Three independent replicates of >100 cells per replicate were quantified. (E) U2OS cells treated with control or γ-tubulin siRNA were coimmunostained with antibodies against γ-tubulin and Centrin1 or γ-tubulin and NEDD1. DNA was stained with DAPI. Scale bars, 5 µm (large images) or 0.5 µm (inset images). (F) Percentages of the cells with different numbers of centrioles and comparisons of those cells with fewer than four centrioles in each treatment in E. Three independent replicates of >100 cells per replicate were quantified. (G) U2OS cells treated with control, γ-tubulin, or NEDD1 siRNA were coimmunostained with antibodies against γ-tubulin and acetylated tubulin. DNA was stained with DAPI. Scale bars, 5 µm (large images) or 0.5 µm (inset images). (H) Percentages of the cells with different numbers of centrioles and comparisons of those cells with fewer than four acetylated-tubulin dots in each treatment in G. Three independent replicates of >100 cells per replicate were quantified. Error bars in B, D, F, and H indicate mean ± SD. ***, P < 0.001 (two-tailed t test). NC, negative control; Tub, tubulin.