Figure 1: Direct interaction of Plk4 with STIL is required for centriolar targeting of STIL and centriole formation.

(a) Co-immunoprecipitation (co-IP) assays testing interactions between the indicated Plk4-FLAG proteins and endogenous STIL. HEK293T cells expressing the empty FLAG vector (–), FLAG-tagged Plk4 full-length (FL) wild-type (FL-WT), FL kinase dead (FL-KD), ΔPEST (Δ272–311 a.a.) WT (ΔP-WT) or ΔPEST KD (ΔP-KD) were immunoprecipitated (IP) with FLAG antibodies. Total cell lysates and IPs were analysed by western blotting using STIL, HsSAS-6, FLAG or tubulin antibodies. (b) Schematic of HA-tagged STIL FL and deletion constructs used for co-IP assays with Plk4ΔPEST-FLAG in HEK293T cells. The right columns show a summary of the co-IP results and centriolar localization of the STIL constructs examined in U2OS cells. The STIL constructs that interact with Plk4ΔPEST-FLAG are represented in red and the minimal binding region in light green. The evolutionarily conserved coiled-coil (CC: a.a. 721–746) and STAN (a.a. 1,061–1,147) domains are indicated. ND, not determined. (c) Alignment of the CC domain within human, mouse, Xenopus and zebrafish STIL and Drosophila Ana2. Identical residues are coloured in yellow; similar residues in grey. Asterisks indicate the residues identical in all aligned sequences; colons: conserved substitutions; periods: semi-conserved substitutions. (d) HEK293T cells co-expressing Plk4ΔPEST-FLAG and HA-STIL FL or STILΔCC were IP with FLAG antibodies. Total cell lysates and IPs were analysed by western blotting using the indicated antibodies. (e) Yeast two-hybrid assay testing interactions between FL or the indicated fragment (a.a. 661–1,017) of STIL and WT or KD Plk4. The empty vectors (–) were used for negative controls. Two independent clones were grown on the plates without histidine and containing 50 mM 3-AT (Supplementary Fig. 1g). (f) U2OS cells were treated with control siRNA (siCnt) or siRNA targeting 3′UTR of endogenous STIL (siSTIL), followed by transfection with an empty vector (vec; –), HA-STIL FL, N-terminal fragment (N), ΔCC or C-terminal fragment (C). The cells were immunostained with antibodies against HA and centrin. DNA is shown in blue. Insets show approximately fivefold magnified views around the centrosome. Scale bar, 5 μm. Histograms represent frequency of interphase cells with centriolar HA (top) or with ≥4 centrin foci (bottom) in each condition. Values are mean percentages±s.d. from three independent experiments (N>50 for each condition). *P<0.05, **P<0.01, NS, not significant (one-tailed t-test).