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. 2019 Aug 8;10:3585. doi: 10.1038/s41467-019-11584-0

Fig. 1.

Fig. 1

Mild replication stress induces whole chromosome mis-segregation. a Mean percentage of γH2AX and 53BP1 positive RPE1 cells. N = 3 independent experiments examining NT = 909, 200 nM Aph = 963, 400 nM Aph = 971 and 1 mM HU = 637 cells; p < 0.0001 in two-way Anova test (yH2AX: NT vs. 400 nM Aph p < 0.0001; NT vs. HU p < 0.0001; 53BP1: NT vs. HU p < 0.0001). b Representatives images of cells stained for γH2AX and DNA after indicated treatments. All scale bars in Figure = 10 μm. c Number of metaphase chromosome breaks. N = 3 examining NT = 222 and Aph = 212 metaphase spreads; p = 0.0008 in one-way Anova test (NT vs. 200 nM Aph p = 0.0114; NT vs. 400 nM Aph p = 0.0005). d Representative image of a Giemsa stained metaphase after a 400 nM Aph treatment; insets show an intact and broken chromosome. e Representative images of RPE1 cells stained for 53BP1 and DNA after indicated treatments. f Percentage of chromosome segregation errors as quantified from time-lapse imaging experiments of RPE1 H2B-mCherry/EB3-GFP cells. N = 3 (Aph) and 6 (NT) examining NT = 551, 200 nM Aph = 423 and 400 nM Aph = 195 anaphases; p = 0.0003 in two-way Anova test (lagging DNA: NT vs. 200 nM Aph p = 0.04; NT vs. 400 nM Aph p = 0.0001). g Representative time-lapse sequence after indicated treatments quantified in (f). Nuclear envelope breakdown (NEBD) is t = 0. White arrow indicates lagging DNA. h Conversion rate of lagging DNA to micronuclei according to the left schematic (created by TW). i Representative time-lapse sequence of cells forming a micronucleus from persistent lagging DNA (white arrow). NEBD is t = 0. j Percentage of interphase cells containing CENP-A-negative and CENP-A-positive micronuclei after indicated treatments. N = 3 examining NT = 2928, Aph = 2421 and Monastrol = 2653. CENP-A positive MN: NT vs. Aph p = 0.0269 in unpaired t-test; NT vs monastrol p = 0.0029. k Representatives image of cells with micronuclei containing (right) or not (left) CENP-A signal; error bars indicate sem. Source data are provided as a Source Data file