Supplementary Figure 2: Common genetic alterations found in CRC and their impact on microtubule dynamic parameters and chromosome number variability.

(a) Genetic alterations commonly found in CRC and their implications in mitotic regulation or chromosome segregation. (b) Representative western blots showing the loss of APC, TP53 or CHK2 or the overexpression of AURKA or PLK1 in HCT116 cells after transfection with siRNAs or cDNAs or in knockout cells, respectively. Since full-length APC is hardly detectable in HCT116 cells, we detected Axin-2/conductin, which is strongly induced on Wnt pathway activation after loss of APC. (c) Representative western blot detecting elevated protein levels of Aurora-A in three independent cell clones derived from HCT116 cells stably overexpressing AURKA. (d) Increased microtubule plus end assembly rates in bipolar mitotic spindles after loss of CHK2. Asynchronously growing HCT116 and isogenic CHK2 knockout cells were subjected to microtubule assembly rate measurements in bipolar mitotic spindles. No significant difference was observed when compared to monopolar spindles measurements (see for example Fig. 2). Scatter dot plots show average assembly rates based on measurements of 20 microtubules per cell (mean ± s.e.m., t-test, n = 30 cells from 3 independent experiments). (e) Karyotype analyses of single cell clones derived from HCT116 cells stably overexpressing AURKA. Single cell clones were grown for 30 generations and the karyotype was determined (100–102 cells analysed per condition). The proportion of cells showing a deviation of the chromosome numbers from the modal within the defined time span was determined as a measure for chromosomal instability. (f) Overexpression of AURKA or loss of CHK2 does not induce supernumerary centrosomes in HCT116 cells. Three independent cell clones overexpressing AURKA or CHK2 knockout cells were investigated for γ-tubulin positive interphase centrosomes. HCT116 cells transiently overexpressing PLK4 serve as a control (mean ± s.d.; n = 2 with a total number of 1,000 cells evaluated). (g–i) No change in other microtubule dynamic parameters in cells with increased microtubule plus end assembly rates. The indicated cell lines expressing EB3-GFP and treated with 67 μM monastrol were live imaged at a rate of one frame per 300 ms. Microtubule tips were automatically tracked using plusTipTracker software package and (g) dynamicity, (h) percent of time spent paused and (i) catastrophe frequency were determined. The graphs show mean values ± SE, t-test (n = 12 cells for each measurement). Detailed data on karyotype analyses can be found in the Supplementary Table 1. Statistic source data for Supplementary Fig. 2 can be found in the Supplementary Table 2.