(a) Centriolar distribution of endogenous Plk4 proteins across the cell cycle. U2OS cells were arrested in prometaphase by treatment with nocodazole for 14 h. The cells were then washed out with fresh media without nocodazole three times and released from the arrest. After the release, the cells were fixed at different phases of the cell cycle and stained with antibodies against Cep152 or STIL as well as Plk4. Insets show ~10-fold magnified views around the centrosome. Scale bar, 5 μm. (b) Quantification of the local signal intensity of centriolar Plk4 and STIL. The local signal intensity was visualized in the indicated colours. The external diameter of centriolar Plk4 rings or dots was measured. Insets show approximately eight-fold magnified views around the centrosome. Scale bar, 5 μm. Values are mean percentages±s.e.m. N, number of total centrosomes. (c) Based on the centriolar distribution of Plk4, centrosomes were placed into two categories (ring-like or dot). The relative representation of each category as well as centriolar STIL over time is shown in the graph. The DNA content of cells for each time point was monitored by flow cytometry (right panels). Values are mean percentages±s.e.m. from four independent experiments for Plk4 (N>60 for each time point) and from two independent experiments for STIL (N>120 for each time point). The schematic represents the typical patterns of centriolar Plk4 and STIL across the cell cycle. (d,e) STIL and HsSAS-6 regulate centriolar distribution of Plk4. (d) U2OS cells were treated with control siRNAs (siCnt) or siRNAs targeting the 3′UTR of HsSAS-6 (siHsSAS-6) or STIL (siSTIL), and stained with antibodies against Plk4 and Cep152. The local signal intensity of centriolar Plk4 was quantified and visualized as indicated (bottom panels). DNA is shown in blue. Insets show approximately ninefold magnified views. Scale bar, 5 μm. (e) Histogram represents percentages of centrosomes categorized based on the centriolar distribution of Plk4. Values are mean percentages±s.e.m. from three independent experiments (N>50 for each condition). For the rescue experiment, U2OS cells treated with siRNAs targeting STIL-3′UTR and expressing HA empty vector (HA), HA-STILΔCC (Δ721–746 a.a.) were stained with antibodies against Plk4 and HA (Supplementary Fig. 7e).