Supplementary Figure 5: Overexpression of AURKA or loss of CHK2 in CRC cell lines results in elevated levels of active centrosomal Aurora-A and increased TACC3 phosphorylation.

(a) Representative western blots showing Aurora-A (left panel) and CHK2 (right panel) protein levels in the indicated CRC cell lines. Note that chromosomally instable CRC cells exhibiting enhanced microtubule growth rates show preferentially either an overexpression of AURKA or a loss of CHK2 correlating with increased microtubule growth rates and chromosomal instability. (b) Overexpression of AURKA causes increased centrosomal levels of active Aurora-A (auto-phosphorylated at threonine-288). Active Aurora-A (pT288) was detected in HCT116 prometaphase cells stably overexpressing AURKA using phospho-specific antibodies in immunofluorescence microscopy experiments. Signal intensities for centrosomal pT288 and normalized to signals obtained for centrosomal centrin are depicted as 3D surface plots and quantified (mean, ±95%-CI, t-test, 26–46 cells as indicated, 3 independent experiments). (c) Increased phosphorylation of TACC3 on overexpression of AURKA in HCT116 cells. Representative western blots showing protein levels of Aurora-A, CHK2, TACC3 and TACC3 phosphorylated at Ser-558 in HCT116 cells stably overexpressing AURKA and synchronized in mitosis (DME 16 h). (d) Increased levels of phosphorylated TACC3 in CHK2 deficient cells. Representative western blots are shown. (e) Determination of the specificity of the antibodies used to detect phosphorylated Aurora-A and TACC3. Representative western blots detecting total and active forms of Aurora-A in HCT116 cells synchronized in mitosis (nocodazole 14.5 h → MG132 1.5 h to prevent mitotic exit) after specific inhibition of Aurora-A (0.5 μM MLN8054) or Aurora-B (2 μM ZM447439). Mitotic indices are shown. (f) Treatment of mitotic cells with MLN8054 abolishes centrosomal signals for active Aurora-A (pT288). HCT116 cells were treated with DME for 3.5 h and subsequently treated with DMSO or 0.5 μM MLN8054 for additional 30 min. Total and active (P-Thr-288) Centrosomal Aurora-A was detected by immunofluorescence microscopy (active Aurora-A, red; total Aurora-A, green; DNA, blue; scale bar, 10 μm). Representative examples of immunofluorescence microscopy pictures are shown. (g) Specificity of the antibodies used to detect the Aurora-A mediated phosphorylation of TACC3. HCT116 cells were transfected with the indicated siRNAs and synchronized in mitosis by DME treatment and subsequently treated with DMSO or 0.5 μM MLN8054. TACC3 and phosphorylated TACC3 as well as total Aurora-A and active Aurora-A were detected on western blots. Representative western blots are shown. Statistic source data for Supplementary Fig. 5 can be found in the Supplementary Table 2.