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. 2019 Nov 28;8:e50292. doi: 10.7554/eLife.50292

Figure 2. DNA damage in isolated micronuclei after mitotic slippage.

(a) Schematic depiction of MMCT. (b) Murine A9 cell before micronucleation. (c) Formation of micronuclei in the A9 donor cell upon colchicine treatment. (d) Microcells with micronucleus isolated via centrifugation. (e) Chromosome painting with probes for chromosome 5 (green) and chromosome 8 (red) in HCT116 before fusion. (f) Micronucleus (yellow arrowhead) next to the primary nucleus in the acceptor cell (HCT116, red arrowhead). (g) Chromosome painting of interphase nuclei in trisomic HCT116. (h) Examples of nucleus of an A9 cell and isolated MN without (-) and with (+) DNA apparent comet. DNA was stained with DAPI. (i) Quantification of cells with a comet in untreated cells (untr), cells treated with colchicine for 48 hr (colch) and in isolated MN. Doxorubicin treated A9 cells were used as a positive control (pos. ctrl). All plots show mean ± s.e.m. of three independent experiments; at least 100 nuclei or MN were scored in each experiment. T-test; p<0.0005. (j) Examples of TUNEL assay in A9 cells treated with colchicine for 48 hr followed by 30 min cytochalasin B (colch+DCB) and in isolated MN. DNA was stained with DAPI. (k) Quantification of TUNEL positive MN under conditions as in (c). All plots show mean ± s.e.m. of at least three independent experiments. N: pos. ctrl = 213, colch = 660, colch+DCB = 137, MN = 2003. T-test; p<0.005. Scale bar: 10 μm.

Figure 2—source data 1. Source data for Figure 2.

Figure 2.

Figure 2—figure supplement 1. Characteristics of micronuclei.

Figure 2—figure supplement 1.

(a) Cells after fusion with isolated MNs were immunolabelled with anti-EdU (red) to detect DNA replication. They were categorized in four groups: non-replicating DNA in primary nuclei (PN) and MN (-/-); replicating DNA in both the PN and MN (+/+); replicating DNA in PN and non-replicating DNA in MN (+/-); non-replicating DNA in PN and replicating DNA in MN (-/+). Representative images of each category were chosen. MNs are marked with arrowheads. DNA was stained with DAPI and Sytox green. RPE1 cells 20 hr after fusion were quantified. Total n: 78. Scale bar: 10 μm. (b) Mean DAPI intensities in TUNEL positive (+) and negative (-) MN. Three independent experiments were performed. N = 280. T-test; p=0.0001. (c) Mean DAPI intensities in MN with NLS and CLS signals and in intact MN. Three experiments, N = 62, T-test; ****p<0.0001. (d) Distribution of lamin B1 positive MN before and after micronuclei isolation. Plots show mean ± s.e.m. of two (before isolation) and one (after isolation) independent experiments. Total N: before = 168; after = 122 MNs. (e) The mean diameter of MN after colchizine treatment quanti_ed in lamin B positive (+) and negative (-) MN visualized with either no (0), 1, 2, 3 or 4 or more (4+) centromeres, three independent experiments. N > 170 MN. (f) _-H2A.x positive MN during a time course of cytochalasin B treatment, without (blue) and with centrifugation (grey). (g) Lamin B1 positive and negative MN were visualized with their diameter (μm) and grouped depending on cytosolic incorporation and DNA damage presence (γ-H2A.x positive in blue and γ-H2A.x negative in grey). Total N: 295 MN. Plots show mean ± s.e.m. of three independent experiments.