Fig. 6

The interaction between CEP85 and STIL is essential for centriole duplication. a The interface residues described in Fig. 5 are required for an efficient interaction of human STIL NTD and CEP85. Western blot showing a pull-down experiment with immobilized, recombinant GST or GST-human STIL NTD, and lysates from cells overexpressing 3xFLAG-tagged CEP85 (WT and mutants) as indicated. b, c IF analysis of CEP85 localization. Tetracycline and hydroxyurea were added to induce the expression of FLAG-CEP85 transgenes and to arrest cells in S-phase for 24 h before fixation. Cells were labelled with the indicated antibodies. Scale bar 10 μm, white boxes indicate the magnified region. c Quantification showing the relative levels of FLAG-CEP85 (WT and mutants) at centrosomes. d Western blot analysis of the indicated protein levels. α-tubulin served as a loading control. e Effect of CEP85 mutations on centriole duplication. U-2 OS cells conditionally expressing FLAG or the siRNA-resistant FLAG-CEP85 (WT and mutants) were treated with control or CEP85 siRNA and induced with tetracycline for 72 h. The G2-phase arrest assays were performed as described in Methods. Quantification showing the percentage of cells with the indicated centriole number (n = 100/experiment, three independent experiments). f, g The role of CEP85 mutations in STIL localization. The S-phase arrest assays (see the Methods) were performed using U-2 OS cells expressing Tet-inducible FLAG or the siRNA-resistant FLAG-CEP85 (WT and mutants) and tetracycline was added for 72 h. Scale bar 10 μm, white boxes indicate the magnified region. g Quantification showing the relative levels of STIL at centrosomes (n = 100/experiment, three independent experiments). h, i Confirming the interaction between CEP85 and STIL in vivo. U-2 OS STIL CRISPR KO cells were co-transfected with GFP-STIL and MAP7-mCherry-CEP85 (WT and mutants) for 24 h. Cells were fixed with 4% PFA and stained with DAPI. i The graph indicates the percentage of cells with STIL recruited to microtubules. j Western blot shows the levels of GFP-STIL and MAP7-mCherry-CEP85 (WT and mutants). Two-tailed t-test was performed for all p-values, all error bars represent SD, and asterisks for p-values are **p < 0.01 and *p < 0.05