Fig 2: NuMA, but not HSET, Aurora A kinase, or TACC3, is upregulated in cyclin D1-overexpressing cells.(A) Western blot of NuMA, TACC3, HSET, Aurora A kinase, and cyclin D1 levels in MCF10APuro (control) and MCF10ACyclin D1 cell lines. The overexpressed cyclin D1 is HA-tagged. Vinculin is shown as a loading control. (B-C) Quantification of NuMA, cyclin D1 (B), HSET, Aurora A kinase, and TACC3 levels (C) normalized to vinculin levels. Lines show mean ± standard deviation of three independent replicates.

Fig 3: E2F1 transactivates stathmin1 and TACC3 in CRC cells. A Schematic representations of the promoter regions of human STMN1 and TACC3. The predicted E2F1 binding sites are indicated. B Agarose gel electrophoresis of ChIP-PCR products in the input, IgG, and ChIP groups, showing that E2F1 interacted with the STMN1 promoter at P3, and with the TACC3 promoter at P2/P3. C Quantitative PCR analysis of the efficiency of interaction of E2F1 DNA at the promoter sites shown in B. D Luciferase activities of DLD1 cells transfected with pGP4.19 promoters carrying wild-type and mutant binding sites promoter, pRL-TK and vector/E2F1

Fig 4: Stathmin1 mutation at Ser16 or depletion of stathmin1 or TACC3 induces the formation of aberrant spindles. A Induction of multipolar and disorganized spindles in DLD1 cells transfected with a plasmid expressing a Ser16-mutated or wild-type stathmin1. After transfection for 48 h, the cells were fixed and stained with antibody to α-tubulin and DAPI. Bar, 10 pixels. B Quantification of the numbers of aberrant spindles in the DLD1 cells described in A. One hundred cells in each group were counted. C Induction of multipolar and disorganized spindles in HCT116 cells transfected with stathmin1, TACC3, or non-specific siRNA. After transfection for 48 h, cells were fixed and stained with antibody to α-tubulin antibody and DAPI. Bar, 10 pixels. D Quantification of the number of aberrant spindles in the HCT116 cells described in C. One hundred cells in each group were counted

Fig 5: Endoplasmic reticulum (ER) stress responses induced by SNIPER(TACC3). (a) SNIPER(TACC3) induces expression of ER stress markers in cancer cells. Cells were treated with 30 μM SNIPER(TACC3)‐2 for 4 h. Whole cell lysates were analyzed by western blotting with the indicated antibodies. (b) XBP1 silencing represses cytoplasmic vacuolization. U2OS cells were transfected with the indicated siRNA for 24 h and treated with 15 μM SNIPER(TACC3)‐2 for 18 h. Phase‐contrast images were obtained (left). Scale Bars: 20 μm. Whole cell lysates were analyzed by western blotting with the indicated antibodies (right). (c) SNIPER(TACC3)‐induced cytoplasmic vacuolation is inhibited by a protein synthesis inhibitor, cycloheximide (CHX) or thiol antioxidants, N‐Acetyl‐L‐cysteine (NAC) and N‐(2‐Mercaptopropionyl)glycine (NMPG). U2OS cells were treated with 50 μg/mL CHX, 20 mM NAC or NMPG in the presence of 30 μM SNIPER(TACC3)‐2 for 5 h. Phase‐contrast images were obtained. Scale Bars: 20 μm. (d) U2OS cells were treated with 20 μg/mL CHX, 10 mM NAC or NMPG in the presence or absence of 20 μM SNIPER(TACC3)‐2 for 24 h and cell viability was measured by WST‐8 cell proliferation assay. The graphs show the means ± SD of a representative experiment performed in triplicate; asterisks indicate P < 0.05 compared with SNIPER(TACC3)‐2 alone.