Supplementary Figure 4: Characterization of cancer-associated Cep152 E21K and V8A mutations impaired in Plk4 binding.

(a–c) The CEP152 E21K mutation found in human colon and rectum adenocarcinoma [Catalogue of Somatic Mutations in Cancer (http://www.sanger.ac.uk/cosmic)] induces a defect in procentriole assembly. U2OS stably expressing either control vector or siRNA-resistant CEP152-sil or CEP152-sil E21K mutant (no tag) were silenced for either control Luciferase (GL siRNA) or CEP152 (CEP152 siRNA). (a) The resulting cells were subjected to immunoblotting analysis using the levels of actin as loading controls. (b,c) The cells in (a) were immunostained with anti-Cep152 and anti-Sas6 antibodies, and the fraction of interphase cells with centrosomally localized Sas6 (b) and the fraction of mitotic cells with abnormal (misaligned and lagging; arrows) chromosome morphologies (c) were quantified from three independent experiments. The results were provided in Figure 3f,g. Representative images used for quantification were shown. Scale bars in (b,c), 10 μm. (d–h) The CEP152 V8A mutation found in human ovarian serous cystadenocarcinoma [Catalogue of Somatic Mutations in Cancer (http://www.sanger.ac.uk/ cosmic)] induces a defect in procentriole assembly and chromosome segregation. (d) 293T cells cotransfected with the indicated constructs were subjected to immunoprecipitation (IP) analyses. Numbers, relative signal intensities. (e) U2OS stably expressing either control vector or siRNA-resistant CEP152-sil or CEP152-sil V8A mutant (no tag) were silenced for either control Luciferase (GL siRNA) or CEP152 (CEP152 siRNA), and then analyzed as in Supplementary Figure 4a–c. (f) The cells in (e) were immunostained with anti-Cep152 and anti-Sas6 antibodies. Representative images are shown. Scale bars, 10 μm. (g,h) From the samples obtained in (f), the number of centrosomal Sas6 signals (from 0 to ≥3) among interphase cells (g) and the number of mitotic cells with aberrant chromosomal morphologies (i.e., misaligned and lagging as shown in Supplementary Figure 4a–c) (h) were quantified from three independent experiments (≥200 cells/cell line/experiment). Bars, s.d.