Figure 3. STIL S428 phosphorylation promotes centriole duplication.

(A) Quantification showing the number of mitotic Centrin foci in cells that were depleted of endogenous STIL and induced to express a siRNA-resistant Myc-GFP-STIL transgene as indicated. Error bars represent the standard error of the mean across three independent experiments. Representative images show example mitotic cells. Scale bars represent 5 µm. (B) The table shows abbreviations for Myc-GFP-STIL transgenes with multiple mutations. ‘PRP’ represents a triple mutation of P404A, R405A, and P408A. (C) Quantification of the relative centrosomal levels of Myc-GFP-STIL constructs from (A) in S/G2 phase cells with at least 40 cells measured per experiment. Bars represent the mean of at least three independent experiments with the average within each experiment shown as a dot. Error bars represent the standard error of the mean. (D) Representative images of data shown in (C). Scale bar represents 5 µm. (E) HEK293FT cells were transfected with the indicated constructs and subjected to co-immunoprecipitation and immunoblotted with the indicated antibodies. Graph represents the mean of relative levels of immunoprecipitated Flag/Myc signal across three independent experiments. A dot displays measurement from each experiment. Error bars represent the standard error of the mean. Asterisks indicate statistically significant differences between measurements (*: p<0.05; **: p<0.005; ***: p<0.0005; ****: p<0.0001). For Figure 3A, statistics were calculated using an unpaired t-test against the fraction of cells containing less than four centrioles in mitosis. For Figure 3C and E, statistics were calculated using a one-sample t-test where mean values were tested as being different from a value of 100.

Figure 3—figure supplement 1. STIL transgenes are expressed to similar levels.

(A) Immunoblot showing the Myc-GFP-STIL transgene expression levels compared to endogenous STIL. Note that a background band (denoted by a red asterisk) is present in the non-transgenic cell lines. (B) Immunoblot showing only Myc-GFP-STIL transgene expression levels after knockdown of endogenous STIL. (C) Experimental outline for the STIL RNAi-replacement experiments.

Figure 3—figure supplement 2. The STIL PRP mutations do not affect STIL S428 phosphorylation.

HEK293FT cells were transfected with the indicated constructs, subjected to co-immunoprecipitation and immunoblotted with the indicated antibodies. (A) Both WT and S428A STIL are capable of activating PLK4 and increasing autophosphorylation of T170. (B) The STIL PRP mutations do not affect S428 phosphorylation.

Figure 3—figure supplement 3. Preventing STIL phosphorylation reduces the accumulation of STIL at the centriole.

Representative images of data shown in (Figure 3C). Scale bar represents 5 µm.

Figure 3—figure supplement 4. Phosphorylation of the STIL STAN motif by PLK4 does not require STIL S428 phosphorylation.

(A) HEK293FT cells were transfected with the indicated constructs, subjected to co-immunoprecipitation and immunoblotted with the indicated antibodies. Preventing phosphorylation of STIL S428 does not block STIL S1116 phosphorylation, and vice-versa. (B) Endogenous STIL was knocked down and replaced with the indicated Myc-GFP-STIL transgene. Where indicated, cells were treated with centrinone for 1 hr. Left, representative images showing Myc-GFP-STIL and pS1108 staining. Scale bar represents 5 µm. Right, quantification of the relative centrosomal ratio of pS1108/GFP-STIL in S/G2 phase cells. Bars represent the mean of three independent experiments in which at least 40 cells were quantified per experiment, and the average within each experiment is shown as a dot. Error bars represent the standard error of the mean. Asterisks indicate statistically significant differences between measurements (*: p<0.05; **: p<0.005; ***: p<0.0005). Statistics were calculated using a one-sample t-test where mean values were tested as being different from a value of 100.