PCNT-CDK5RAP2 and centrioles act in parallel to direct spindle assembly and chromosome segregation. (A and B) Analysis of relative proliferation (A) and centrosome number measured by counting γ-tubulin foci (B) following 4-d mock (DMSO [D]) or centrinone (C) treatment. Scale bar, 10 µm. (C) Live imaging of mitosis for the indicated conditions using the procedure on the left. Cells were first treated with DMSO (D) or centrinone (C) to generate centriole-less cells, labeled with SiR-DNA for 2 h, and then imaged at 5-min intervals. Mitotic outcomes were classified into four categories, representative examples of which are shown in the image series. Note that the “Normal Segregation” category images are from the Control sequence in Fig. 1 C. Numbers on the top left of image panels indicate time after NEBD in minutes. Colored squares to the right indicate the labeling scheme for each outcome. Scale bar, 5 µm. (D and E) Quantification of mitotic outcomes (D) and mitotic duration (E) for the indicated conditions. Error bars in E are the 95% CI. (F) RNAi analysis. Left: Immunoblot of indicated conditions. α-tubulin is a loading control; numbers below indicate relative total band intensities. Right: Quantification of nuclear morphology as a readout of mitotic outcomes. Mean and SD from three independent experiments are plotted. At least 200 nuclei were scored per condition per experiment. P values are from t tests. (G) Mitotic spindle morphology for the indicated conditions; for detailed protocol, see Fig. S2 A. Arrow marks extra microtubule focus frequently observed in centrinone-treated cells. Fixed cells were stained to label microtubules and DNA. Scale bar, 10 µm. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. IB, immunoblot; IF, immunofluorescence; ns, not significant; tub, tubulin.