PCNT and CDK5RAP2 are dispensable for spindle assembly and chromosome segregation in RPE1 cells. (A) Top: Images of fixed mitotic RPE1(USP28Δ) cells lacking PCNT or CDK5RAP2 after labeling with the indicated antibodies and a DNA dye. Scale bar, 10 µm. Bottom: Immunoblots analyzing CDK5RAP2 and PCNT in the parental line and knockout clones. α-tubulin is a loading control. (B) Effect of PCNT or CDK5RAP2 loss on localization of CEP192. Scale bar, 5 µm. (C) Live imaging of mitosis in cells lacking PCNT or CDK5RAP2. Cells were labeled with SiR-DNA for 2 h and then imaged at 5-min intervals. Quantification of mitotic duration is shown below for 20 cells per condition. Error bars represent SD. Scale bar, 10 µm. (D and E) CDK5RAP2 depletion removes residual centrosome-localized CDK5RAP2 in PCNTΔ cells. (D) Images of fixed cells for the indicated conditions; 2.5× magnified regions to the right show CDK5RAP2 signal in the centrosome region. Scale bar, 10 µm. (E) Centrosomal CDK5RAP2 intensity for the indicated conditions. Error bars represent SD. (F) Mitotic spindle morphology, 48 h after siRNA transfection, for the indicated conditions. Fixed cells were stained to label microtubules and DNA. Spindle morphology in prometaphase and metaphase stage cells was scored. MT, microtubules; ns, not significant; tub, tubulin. ****, P < 0.0001; ns, not significant (P > 0.05).