Figure 3. The STIL-CC motif is essential for centriole duplication.

(A) Immunofluorescence microscopy of U2OS cells transfected with STIL-WT, STIL-ΔCC or STIL-ΔSTAN for 48 hr. Cells were fixed and stained with the indicated antibodies. Scale bar denotes 1 µm. (B) Quantification of centriole numbers in U2OS cells after overexpression of the indicated STIL plasmids (3 experiments, a total of 300 cells were analyzed for each condition). Error bars denote SD. (C) Scatter plot to illustrate STIL signal intensity at centrosomes, after overexpression of STIL-WT, STIL-ΔCC or STIL-ΔSTAN (20 centrosomes were analyzed for each condition). (D–F) 3D-SIM images of U2OS cells that have been transfected with EGFP-tagged STIL-WT, STIL-ΔCC and STIL-ΔSTAN and stained with the indicated antibodies. (G) Scatter plot to illustrate measured PLK4 signal intensities at centrosomes, after overexpression of STIL-WT/ΔCC or ΔSTAN (60 centrosomes were analyzed for each condition). Scale bar denotes 1 µm.

DOI: http://dx.doi.org/10.7554/eLife.07888.007

Figure 3—figure supplement 1. The STIL-CC domain is essential for STIL oligomerization.

(A) Schematic illustration of STIL constructs used for the co-immunoprecipitation experiments shown in (B–D). On the right, the relative strengths of the interactions are indicated (+, strong; ±, weak; -, not detected). (B–D) Western blot analysis of co-immunoprecipitation experiments to map the region required for STIL self-association. HEK293T cells were transfected with the indicated plasmids to co-express the corresponding STIL constructs for 24–36 hr. Subsequently, cells were lysed and co-immunoprecipitations were performed with anti-myc or anti-FLAG antibodies.

DOI: http://dx.doi.org/10.7554/eLife.07888.008