Restoration of Plk4 levels in acentriolar cells leads to de novo centriole formation. (A) Immunoblot showing the level of immunoprecipitated endogenous Plk4-AID-FLAG at the indicated times after IAA washout in acentriolar, Plk4^AID/AID;p53 shRNA cells. (B) Quantification of the number of Centrin foci per interphase cell at indicated times after IAA washout, in Plk4^AID/AID;p53 shRNA cells. Bars represent the mean of >200 cells from three independent experiments. (C, top) Quantification of the number of Centrin-marked de novo centrioles in interphase cells at 2 d after IAA washout (WO). Cells were transfected with the STIL siRNA 24 h before IAA washout. (bottom) Immunoblot showing depletion of STIL at 48 h after transfection with STIL siRNA. (D and E) Quantification of the fraction of bipolar and multipolar divisions in Plk4^AID/AID;p53 shRNA at indicated times after IAA washout. Bars represent the mean of >120 cells from two independent experiments. All error bars in the figure represent the SEM. (F) Selected images of anaphase phenotypes. Both single and pairs of de novo centrioles clustered at the poles of the mitotic spindle. Cells were costained with Centrin and CEP192. (G) Selected images of Centrin-marked de novo centrioles at 1 d after IAA washout. Cells were costained with Plk4, STIL, SAS6, CPAP, CEP192, CEP152, and CEP135. Bars: (main) 5 µm; (inset) 1 µm.