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. 2018 Aug 6;7:e36898. doi: 10.7554/eLife.36898

Figure 6. DNA intercalation compromises SAC response by interfering with Aurora B localization.

Figure 6.

(A) Normalized expression of Mcl1 in asynchronous control HeLa cells or cells treated with ethidium bromide (EtBr) (EtBr, 0.71 ± 0.06, median ±SD from three technical replicates, n.s. p>0.05, Mann-Whitney Rank Sum Test). (B) Scattered plot showing the duration of the mitotic arrest of control (NOC) and Hela cells treated with ethidium bromide (NOC + EtBr) after nocodazole treatment (NOC, 19.2 ± 7.0 hr, n = 386; NOC + EtBr, 18.1 ± 5.9 hr, n = 151; median ±SD; n.s. p>0.05, Mann-Whitney Rank Sum Test). The red line represents the mean and the error bars represent the standard deviation from a pool of at least two independent experiments. (C) Cell fate of mitotic HeLa cells treated with the same drugs as in 5B. (D) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or ethidium bromide for 4 hr (NOC + EtBr) after nocodazole treatment with the indicated antibodies. Scale bar = 5 µm. 10x magnification of a pair of kinetochores are shown in the right. Dashed circle encompasses a single kinetochore and the arrow indicate the position of centromeric Aurora B. Scale bar = 0.5 µm. (E) Normalized ratio of pAurora B/ACA fluorescence signal at inner centromere of NOC and NOC + EtBr. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation. (NOC, 1.00 ± 0.48, n = 474; NOC + EtBr, 0.79 ± 0.23, n = 143; ****p≤0.0001 relative to control, Mann-Whitney Rank Sum Test). (F) Shows the Electrophoretic Mobility Shift Assay (EMSA) experiments carried out to assess the binding chromosomal passenger complex (CPC) ability to recombinant nucleosome core particles (NCPs) in the presence of DMSO, ActD, Triptolide or EtBr. Different molar concentrations (0, 25 and 50 nM) of CPC were tested. (G) The bar diagram shows the densitometric profile of the autoradiograph depicted in F. The bar represents the mean ±SD from at least three independent experiments (n.s. p>0.05, *p≤0.05 relative to control, t test).