(a,b) U2OS cells were arrested in G1/S phase by treatment with aphidicolin (Aph) for 18 h, followed by addition of 10 μM MG132 (±) for 6 h. Cells were then fixed and stained with antibodies against Plk4 and STIL. DNA is shown in blue. In this figure, all insets show approximately ninefold magnified views around the centrosome. Scale bars, 5 μm. The local signal intensity of centriolar Plk4 and STIL was quantified and visualized as indicated. The percentages of centrosomes with Plk4 rings or Plk4 dots were quantified in b (N≥40 centrosomes with STIL from four independent experiment). Similar experiments were also performed with U2OS cells in G1/S phase using cell cycle synchronization (sync.) with nocodazole (Noc) treatment in b. (c) U2OS cells treated with siRNAs targeting STIL-3′UTR (siSTIL) and expressing HA-STIL full length at low or high levels, ΔSTAN (Δ1,061–1,147 a.a.), 5A non-phosphorylatable mutant, PACT-full length or PACT-ΔCC were stained with antibodies against Plk4 and HA. Note that the signal intensity of centriolar Plk4 ring in the absence of endogenous STIL or HsSAS-6 is relatively higher than that in the case of overexpression of STIL full length or mutants used in this experiment. The experiment was repeated at least three times. (d) Ubiquitination assay. HEK293T cells transfected with the indicated combination of plasmids were treated with 10 μM MG132 (±) for 6 h. The cells were then immunoprecipitated (IP) with FLAG antibodies. Total cell lysate and IPs were analysed by western blotting using Myc, FLAG, STIL or tubulin antibodies. (e) HEK293T cells co-expressing Plk4ΔPEST-FLAG wild-type or the kinase dead (KD) and HA-STIL full length or STILΔN3 were IP with FLAG antibodies. Total cell lysate and IPs were analysed by western blotting using STIL, FLAG or tubulin antibodies. The values on the bottom indicate the relative amount of IP Plk4ΔPEST-FLAG. Values are mean percentages±s.e.m. from three independent experiments.