Examples of small CEP192 foci in PLK1-inhibited acentriolar mitotic cells and analysis of induced CEP192 knockout. (A) Example of small CEP192 foci observed in PLK1-inhibited acentriolar cells. Two examples are shown. Note that control cell image shown is 60 min after NEBD; the left PLK1-inhibited cell is 95 min after NEBD and the right PLK1-inhibited cell is 40 min after NEBD. Scale bar, 10 µm. (B) Top: Schematic of experimental approach. The inducible CEP192 knockout cell line was described previously (Meitinger et al., 2020). Cells were first treated with centrinone, and then CEP192 was inducibly deleted by adding doxycycline to express Cas9. Cells were then fixed and processed for immunofluorescence. Middle and bottom: Analysis of CDK5RAP2 (middle) and PCNT (bottom) localization in the indicated conditions. Note that the centrinone-only condition is the iCEP192 knockout cell line treated with centrinone but not with doxycycline. Scale bar, 10 µm. (C) Quantification of metaphase frequency (left) and acentriolar foci frequency (right) in the indicated conditions. Metaphase alignment is greatly reduced in frequency in centrinone-treated mitotic cells in which CEP192 has been knocked out; thus, the quantification of acentriolar foci only employed prometaphase stage cells from the control. Note that by metaphase, essentially all centrinone-treated control cells have robust acentriolar foci at their spindle poles (e.g., see Fig. S5 A). Dox, doxycycline; KO, knockout.