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. 2016 Nov 21;7:13508. doi: 10.1038/ncomms13508

Figure 3. No colocalization of Cep152 with extra Cep63 dots.

Figure 3

(a) Wild-type MEFs were transfected with dKeima–Cep63 and GFP–SAS6. Magnified images are shown at the upper left and lower right corners. White lines indicate the cell shape. (b–d) Atg5−/− MEFs were transfected with siCep63 and scramble siRNA (siCtrl) for 24 h and were immunostained with anti-γ-tubulin antibody. (b) Quantitative PCR confirmed the reduction of Cep63 mRNA. (c) The number of extra Cep63 dots per cell was calculated (n>30 cells). (d) The percentage of cells with three or more centrosomes is shown. (e,f) WT MEFs were transfected with Flag–Cep63 and a control vector for 24 h. (e) The expression of Flag–Cep63 was confirmed by western blotting. (f) The percentage of cells with three or more centrosomes is shown. (g) No colocalization of Cep152 with extra Cep63 dots in the Atg5−/− MEFs. Arrow and arrowhead indicates mature centrosome and extra Cep63 dot, respectively. Magnified images are shown at the right. (h) No difference of extra Cep152 dots between WT MEFs and Atg5−/− MEFs. Arrows and arrowheads indicate mature centrosomes and extra Cep152 dots, respectively. (i) The number of extra Cep152 dots per cell was calculated (n>30 cells). (j) No influence of Cep152 silencing on extra Cep63 dots. Atg5−/− MEFs were transfected with siCep152 and siCtrl for 24 h. Arrows and arrowheads indicate mature centrosomes and extra Cep63 dots, respectively. Scale bar, 5 μm. (k) The number of extra Cep63 dots per cell was calculated (n>30 cells). (l) Lysates from MEFs expressing GFP–Cep63 and Flag–Cep63 were immunoprecipitated with anti-Flag antibody and normal IgG. ‘Total' indicates 5% of the lysates subjected to immunoprecipitation. In e and l, uncropped images are shown in Supplementary Fig. 14. Scale bar, 10 μm (a,g,h). In b,d and f, data are mean+s.d. (n=3). In c,i and k, the red lines indicate mean values. In b–d and f, *P<0.05, Student's t-test. In i and k, ‘NS' indicates no significant difference ((i) analysis of variance (ANOVA) Tukey's post hoc test, (k) Student's t-test).