Fig 2: Deletion of TACC3 C-terminal (TACC) domain affects ?-tubulin localization but not TACC3–?-TuRC interaction(A) Schematic representation of GFP-TACC3 (WT) and GFP-TACC3 ?C (1–590) constructs. Position of Ser 558 phosphorylation site is also shown. (B) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT) along with TACC3 3'-UTR siRNA and GFP-TACC3 ?C (1–590) along with TACC3 esiRNA for >48 h were analyzed by Western blot to detect the levels of exogenous TACC3 proteins and the depletion of endogenous TACC3 by the respective siRNAs. Both endogenous TACC3 and the exogenous TACC3 proteins were probed with mouse monoclonal anti-TACC3 antibody. a-tubulin was probed as a control. (C) Representative confocal images of GFP-TACC3 (WT) and GFP-TACC3 ?C (1–590) transfected mitotic HeLa Kyoto cells showing the differences in ?-tubulin intensity. Scale bar, 5 µm, (D) The plots show ?-tubulin intensity at the centrosomes in different conditions as indicated. Centrosomal ROI used for quantification for both cases is shown. (E) The bar graph shows the percentage of cells (cells with above the average centrosomal ?-tubulin intensity of TACC3 WT-expressed cells) with decreased ?-tubulin intensity phenotype in GFP-TACC3 ?C cells. The bars represent mean ± S.E. The number of mitotic cells counted = 60 each (four independent experiments). ****, P<0.0001, ***, P<0.001. (F) Immunoprecipitation of GFP-tagged TACC3 ?C using the anti-GFP antibody in the lysate of HEK293T cells transfected with GFP-TACC3 ?C. The immunoblots were probed with GFP-TACC3 ?C, ?-tubulin, GCP3, GCP4, and GCP6 using the respective antibodies. (G) Immunoprecipitation of ?-tubulin using anti-?-tubulin antibody in the lysate of HEK cells transfected with GFP-TACC3 ?C. The immunoblots were probed with GFP-TACC3 ?C and ?-tubulin using the respective antibodies. (H) Immunoprecipitation of GCP4 using anti-GCP4 antibody in the lysate of HEK293T cells transfected with GFP-TACC3 ?C. The immunoblots were probed for GFP-TACC3 ?C along with GCP4 by using respective antibodies.

Fig 3: Perturbation of TACC3–ch-TOG interaction stimulates recruitment of ?-TuRC proteins to centrosomes(A) Schematic representation of GFP-TACC3 (?678–681)-TACC3 shRNA and GFP-TACC3 (?682–688)-TACC3 shRNA constructs. (B) Lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (?682–688)-TACC3 shRNA, or GFP-TACC3 (?678–681)-TACC3 shRNA for 48 h were analyzed for exogenous TACC3 proteins with simultaneous depletion of endogenous TACC3. Both endogenous TACC3 and GFP-TACC3 proteins were stained with mouse monoclonal TACC3 antibody. Actin was probed as a control. TACC3 level in control untrasfected cells is also shown. (C) Immunoprecipitation of GFP-tagged TACC3 proteins using the anti-GFP antibody in the lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (?678–681)-TACC3 shRNA, or GFP-TACC3 (?682–688)-TACC3 shRNA. The immunoblots were probed with GFP and ch-TOG antibodies. (D) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (?678-681)-TACC3 shRNA (middle panel), or GFP-TACC3 (?682–688)-TACC3 shRNA (bottom panel)- transfected mitotic HeLa Kyoto cells showing the differences in ?-tubulin levels at the centrosomes. Scale bar, 5 µm, ?-tubulin, and ch-TOG were stained with mouse monoclonal anti-?-tubulin and rabbit polyclonal anti-ch-TOG antibody, respectively. (E) The bar graph shows the quantification of ?-tubulin intensity at the centrosomes (two) in different conditions as of (d). (F) Representative confocal images of GFP-TACC3 (WT)-TACC3 shRNA (top panel), GFP-TACC3 (?678–681)-TACC3 shRNA (middle panel), or GFP-TACC3 (?682–688)-TACC3 shRNA (bottom panel) transfected mitotic HeLa Kyoto cells showing the differences in GCP6 localization at centrosomes. Scale bar, 5 µm, GCP6 was stained with mouse monoclonal anti-GCP6 antibody. (G) The bar graph shows the quantification of GCP6 intensity at the centrosomes in different conditions as in panel (F). (H) The bar graph shows the percentage of cells (cells with above the average centrosomal ?-tubulin intensity of TACC3 WT-expressed cells) with increased ?-tubulin intensity phenotype in both the mutant cases. The bars in (e), (g), and (h) represent mean ± S.E. The number of mitotic cells counted = 100 each (four independent experiments); ****, P<0.0001, ***, P<0.001.

Fig 4: Inhibition of TACC3–ch-TOG binding enhances interaction of TACC3 with ?-TuRCs(A) Immunoprecipitation of GFP-tagged TACC3 proteins using GFP antibody in lysates of HeLa Kyoto cells transfected with GFP-TACC3 (WT)-TACC3 shRNA, GFP-TACC3 (?678–681)-TACC3 shRNA, or GFP-TACC3 (?682–688)-TACC3 shRNA. The immunoblots were probed with GFP-TACC3 (WT), GFP-TACC3 (?678–681), and GFP-TACC3 (?682–688) forms along with ?-tubulin, GCP3, GCP4, and GCP6 by using the respective antibodies. (B) Fold change of different ?-TuRC proteins from the GFP-TACC3 immunoprecipitates of GFP-TACC3 WT, GFP-TACC3 (?678–681), or GFP-TACC3 (?682–688) expressed cells are plotted (based on three experiments in each). Data are mean ± S.E. ****, P<0.0001, ***, P<0.001, **, P<0.01, ns: not significant.