Mitotic chromosome misalignment is independent of DNA replication.

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Mitotic chromosome misalignment is independent of DNA replication. Mitotic chromosome misalignment is independent of DNA replication. (A) DNA is replicated in cycled but not CSF extracts. To assemble cycled spindles, sperm was incubated in interphase extract and cycled to mitosis in the presence of Cy3-labeled dUTP. To assemble CSF spindles, sperm was incubated in CSF extract in the presence of Cy3-labeled dUTP. (B) Chromosome misalignment in RECQL4-depleted CSF extracts. To assemble CSF spindles, sperm was incubated in CSF extract in the presence of Cy3-labeled tubulin. Samples were fixed, stained with DAPI, and analyzed by confocal microscopy. The frequency of bipolar spindles was counted taking all chromatin structures into account. Chromosome alignment was quantified analyzing all bipolar spindle structures identified. Columns show the average of three independent experiments and circles indicate individual data points. (C) RECQL4 depletion delays but does not prevent DNA replication. Sperm was incubated in interphase extract in the presence of Cy3-labeled dUTP. Samples were fixed at 45 and 90 min, stained with DAPI, and analyzed by confocal microscopy. dUTP intensity on chromatin was quantified using image J. Error bars represent SD. n > 20 structures, N = 2 experiments. ****P < 0.0001; NS (not significant) P > 0.05 (t test, two-tailed). Scale bars, 20 μm. Hideki Yokoyama et al. LSA 2019;2:e201800120 © 2019 Yokoyama et al.