Extended Data Fig. 9. Characterization of MN-bodies.
(a) Same-cell live-fixed experiment supporting the fixed imaging shown in Fig. 3a, b. Top, scheme of the experiment. MN were induced in RPE-1 cells and the MN fate was tracked with GFP-H2B and RFP-NLS (to visualize MN NE rupture in generation 1). After most cells progressed into generation 2, they were fixed and labeled to detect γH2AX. Bottom left: representative images of a daughter cell pair, one with and one without an MN-body. Bottom right: summary of 13 cell pairs tracked and analyzed by the same-cell live-fixed experiments (from two experiments). Scale bars 5 µm. (b) MDC1 accumulation on a mitotic chromosome in an RPE-1 cell that had a micronucleus in the prior interphase (generated by nocodazole block and release), shown by immunofluorescence staining of endogenous MDC1 (representative images from two experiments). Note that the micronuclear chromosome can be identified because it is decondensed, a known feature of mitotic micronuclear chromosomes. Scale bar 5 µm. (c) Images from a timelapse series tracking damaged MN chromosomes through cell division and MN-body formation. GFP-H2B: chromosomes; green arrowheads: MN chromosome; RFP-NLS: NE integrity; blue arrowheads: MN NE rupture; red arrowheads: SNAP-MDC1-marked MN DNA damage. Time: hours post release from the nocodazole block for MN induction. Scale bars 5 µm. (d) Durations of MN-bodies assessed by live-cell imaging of SNAP-MDC1 indicate MN-bodies persist throughout most of the generation 2 interphase. Each row shows the lifetime of a MN-body (black bar) and the duration of imaging (light grey bar). In all but four cases, the MN-bodies persisted until the end of the imaging (see Extended Data Fig. 9c for an example of a time lapse series). Note that analysis of the live-cell imaging experiments showed that 68% of cells with MN-bodies were derived from mother cells with a micronucleus that ruptured, 22% were derived from mother cells with intact micronuclei and 10% from non-micronucleated mother cells. (e) Distribution of signal intensities for MN-body by immunofluorescence staining for the endogenous MDC1. Performed and analyzed as in Fig. 3c (n = 341, from two experiments). Median with 95% CI. Two-tailed Mann–Whitney. (f) Determination of the background nuclear RNAP2-Ser5ph signal in nucleoli. We measured the background RNAP2-Ser5ph signal in nucleoli (fibrillarin positive), which should lack active RNA polymerase II, and in nuclear regions lacking nucleoli. These values were then normalized to the density of fluorescence intensity from a nuclear mask excluding the nucleoli. The detection of measurable RNAP2-Ser5ph signal in the nucleoli means that we likely underestimate the extent of RNAP2-Ser5ph signal loss in MN-bodies (see Methods; n = 650, from two experiments). Median with 95% CI. Kruskal-Wallis with Dunn’s multiple comparisons test. (g) Verification of low transcription and H3K27ac loss in MN-bodies in U2OS cells. Performed and analyzed as in Fig. 3c (n = 138, from two experiments). (h) Reduced H3K9ac (left) but not H3K9me2 (middle) or H3K27me3 (right) in MN-bodies. Performed and analyzed as in Fig. 3c (n = 222, 234 and 244, left to right, from two experiments). (i) H3S10ph and H3T3ph levels show no increase but a minor decrease in MN-bodies compared to the control. Performed and analyzed as in Fig. 3c (n = 130 left; n = 124 right, from two experiments).