Extended Data Fig. 10: DamMN system characterization.
From: Heritable transcriptional defects from aberrations of nuclear architecture
(a) Validation of the DamMN system. Shown are representative single-focal plane confocal images of RPE-1 megaDam cells ~45 h post release from the CDK1-induced G2 block at the start of the experiment (see Fig. 4a and Methods). There is no m6A DNA methylation if megaDam transcript is not induced (left, no Dox); if megaDam is not degraded prior to mitotic entry, all primary nuclei show m6A DNA methylation because of labeling during mitosis (middle, Dox, no ASV no IAA); if megaDam is degraded prior to mitosis because of size exclusion through the NE by passive import, primary nuclei are mostly not m6A methylated (right, Dox, +ASV +IAA). m6A methylation is visualized with the m6A-Tracer (see Fig. 4a and Methods; four experiments). Scale bars 20 µm. Note that even in the condition of degrading megaDam before mitosis (Dox, +ASV +IAA), many cells still show whole nucleus labeling with the m6A-Tracer. This could either result from cells that were in mitosis at the time of megaDam induction or from cells where nuclear exclusion of megaDam was not complete. (b) Efficient induction and degradation of megaDam. FACS analysis to detect mCherry-tagged megaDam. All samples are unsynchronized RPE-1cells with or without megaDam, with or without megaDam transcriptional induction or megaDam degradation for the indicated periods of time. The controls are RPE-1 cells lacking the megaDam construct showing no background autofluorescence without or with Dox treatment. Shown is the percentage of cells expressing mCherry (PE channel, from two experiments). (c) Western blot to detect megaDam for the indicated samples corresponding to the experiment shown in (b), above. Shown is a cropped image of a gel from the region at the megaDam molecular weight (~130 kDa). Note: the a-mCherry Ab detects non-specific background bands, but megaDam is readily distinguished from these background bands (two experiments). * indicates a background band. For gel source data, see Supplementary Fig. 1. (d) Specific labeling of MN chromosomes in mitotic cells (two examples) using the DamMN system. Top shows an MN chromosome in a prometaphase cell lacking DNA damage. Bottom shows an MN chromosome in a metaphase cell with DNA damage. Note that the MN chromosome is less condensed during mitosis, as has been previously described62. Performed as described in Fig. 4a. Yellow dashed line: an MN chromosome positive for γH2AX and m6Tracer (n = 4 experiments). Scale bars 5 µm. (e) Control for Fig. 4c showing the distribution of MN-body γH2AX FI units relative to the general nuclear background (lacking nucleoli). The MN-body region of interest corresponds to the m6A-Tracer signal (see Methods). The γH2AX low MN-bodies were designated if the total area of γH2AX positive pixels (>3SD above background, see Methods) occupy less than 21% of the MN-body area (corresponding to the bottom quartile of γH2AX positive MN-bodies). The designation of γH2AX intermediate MN-bodies was between 21% and 65.7% of the MN-body area (the middle two quartiles), and γH2AX high was >65.7% of the MN-body area (the top quartile of MN-bodies) (left to right: n = 220, 111, 220 and 112, four experiments). Error bars: median with 95% CI.
