Analysis of spindle assembly following recovery from nocodazole treatment of synchronized cells and results of acute centrinone treatment. (A) Schematic of protocol used to synchronize cells, release into nocodazole, and recover from nocodazole before fixation and immunofluorescence. siRNA transfection was used to deplete CDK5RAP2 or PCNT in order to assess the impact on spindle assembly. (B) Analysis of acute centrinone treatment of CDK5RAP2- and PCNT-depleted cells. Centriole loss following centrinone treatment requires cell division; thus, in the first 24 h after addition of centrinone, PLK4 kinase activity is inhibited but centrioles are still present. Acute centrinone treatment did not result in a synthetic mitotic defect in combination with CDK5RAP2 or PCNT depletion. Ctrl, control.