Supplementary Figure 4: Increased microtubule assembly rates and impaired error correction are separable mechanisms.

(a) Determination of microtubule turnover. Examples of time-lapse fluorescent images of HCT116 spindles before (−6.0 s) and after photo-activation of GFP-tubulin. Scale bar, 6 μm. (b–c) Normalized fluorescence intensity over time after photo-activating spindles of HCT116 cells with and without AURKA overexpression or loss of CHK2 (mean ± s.e.m., n = 13–36 cells as indicated). (d) Quantification of the proportion of lagging chromosomes in HCT116-CHK2^−/− cells with and without siRNA-mediated knockdown of MCAK after monastrol washout and after release from monastrol into MG132 to prolong the time for error correction (mean ± s.d., t-test, n = 4 independent experiments with a total number of 400 cells evaluated). (e) Representative western blots showing the loss of MCAK in HCT116 cells after transfection with siRNAs. (f) Repression of MCAK does not alter microtubule plus end assembly rates. HCT116 cells were transfected with a siRNA targeting MCAK and microtubule plus end assembly rates were determined. Scatter dot plots show average growth rates based on measurements of 20 microtubules per cell (mean ± s.e.m., t-test, n = 20 cells from 3 independent experiments). (g) Representative western blots showing protein levels for MCAK in different CRC cells lines overexpressing human MCAK. (h) Overexpression of MCAK in CIN cells does not alter microtubule plus end assembly rates. Different CRC cells overexpressing MCAK were used to determine microtubule plus end assembly rates. Scatter dot plots show average growth rates based on measurements of 20 microtubules per cell (mean ± s.e.m., t-test, n = 20 cells from 3 independent experiments). i, Determination of microtubule plus end assembly rates in HCT116 cells after monastrol washout and after release from monastrol into MG132. Scatter dot plots show average microtubule assembly rates based on measurements of 20 microtubules per cell (mean ± s.e.m., t-test, n = 10, cells from 2 independent experiments). (j), Determination of kinetochore-microtubule turnover. Normalized fluorescence intensities over time after photoactivation of spindles in HCT116 cells expressing PA-GFP-tubulin immediately after establishing bipolar spindles on release from monastrol (early) and after release from monastrol into MG132 (late) (mean ± s.e.m., n = 14–33 cells as indicated at 33 time points each). Statistic source data for Supplementary Fig. 4 can be found in the Supplementary Table 2.