Supplementary Figure 7: Partial loss of AURKA in CRC cell lines restores normal mitotic progression, kinetochore microtubule turnover and chromosomal stability without affecting normal cell cycle progression.

(a) Representative western blots showing reduced protein levels of Aurora-A in HCT116-CHK2^−/− cells stably expressing AURKA shRNAs. (b) Representative western blots showing reduced phosphorylation of TACC3 (P-Ser-558) in HCT116-CHK2^−/− cells after partial loss of AURKA. (c) Partial loss of AURKA in CHK2 deficient cells does not affect cell cycle progression. Stable cell lines expressing shRNAs targeting AURKA were subjected to FACS analyses. Representative cell cycle profiles are shown. (d) Partial loss of AURKA in HCT116-CHK2^−/− cells restores normal mitotic timing. The time from nuclear envelope breakdown (NEB) to anaphase onset was determined by live cell microscopy. The box and whisker plot shows the range, median and quartile of the measurements (t-test, n = 59–163 cells as indicated, 3 independent experiments). (e) Partial loss of AURKA suppresses hyper-stable kinetochore-microtubule attachments in cells with increased microtubule assembly rates. The indicated cell lines expressing photo-activatable GFP-tubulin (PA-GFP-tubulin) were used to determine kinetochore-microtubule turnover and a summary of the measurements is given (s.e.m., n = 8–9 cells as indicated, 3 independent experiments). (f) Determination of kinetochore-microtubule turnover. The graph displays normalized fluorescence intensities over time after photoactivation of spindles in HCT116-CHK2^−/− and cells expressing shRNAs targeting AURKA clone 1 (mean ± s.e.m., n = 8–9 cells as indicated, 3 independent experiments). (g) Karyotype analyses of HCT116-CHK2^−/− cells after partial repression of AURKA using CEP-FISH. Single cell clones derived from HCT116-CHK2^−/− cells expressing control or AURKA shRNAs were grown for 30 generations and subsequently subjected to CEP-FISH analysis. The proportion of cells that deviate from the modal chromosome number of chromosome 7 and chromosome 15 were calculated (100 cells analysed per condition). (h) Representative western blots showing the protein levels of Aurora-A and phosphorylated TACC3 (P-Ser-558) in various CRC cell lines stably expressing control or shRNAs targeting AURKA. i, Karyotype analyses of SW620 cells after partial repression of AURKA using CEP-FISH. Single cell clones derived from SW620 cells expressing control or AURKA shRNAs were grown for 30 generations and subsequently subjected to CEP-FISH analysis. The proportion of cells that deviate from the modal chromosome number of chromosome 7 and chromosome 15 were calculated (100 cells analysed per condition). Detailed data on karyotype analyses can be found in the Supplementary Table 1. Statistic source data for Supplementary Fig. 7 can be found in the Supplementary Table 2.