Fig 2: Gene expression from foreign DNA occurs after mitosis.a Schematic of the experiment. Top: diagram of pLacO-pEF1α-RFP. This plasmid carries a lacO repeat sequence (256 repeats, approximately 10 kbp) and a sequence expressing a monomeric red fluorescent protein (RFP) under the EF1α promoter. Bottom left: when the transfected plasmid is exposed to the cytosol upon endosome rupture, it binds to GFP-LacI and becomes fluorescent. Bottom right: when it enters the nucleus, the expression of RFP occurs. Therefore, RFP fluorescence is an indication of the nuclear translocation of the plasmid. While using this system, exogenous gene expression was determined by the appearance of RFP fluorescence. b HeLa/GFP-LacI cells transfected with pLacO-pEF1α-RFP (0.5 μg/dish) diagrammed in a. Immediately after transfection, the cells were fixed and stained using anti-BAF or anti-emerin antibodies. DNA (stained with DAPI, blue), GFP-LacI (green), and BAF (red) or emerin (red) in the merged image. Arrows indicate DAPI signals with GFP-LacI signals. Arrowhead indicates DAPI signals without GFP-LacI signals. The inset shows an image with increased brightness in the boxed region. Bar, 10 μm. c Time-lapse live-cell images of transfected HeLa/GFP-LacI cells. DNA was stained with Hoechst 33342 to determine the timing of chromosome segregation. Immediately after transfection, acquisition of time-lapse images at 10-min intervals was started. Selected time frames are shown. Images at each time frame are maximum intensity projection images for z-stacks. The numbers represent time (h:min). Time 0 represents the onset of chromosome segregation. Merged images, DNA (Hoechst 33342, white) and RFP (magenta). Bar, 20 μm. d Timing of protein expression from transfected DNA. The timings of RFP expression and chromosome segregation were determined in individual cells by time-lapse live-cell imaging as described in c. X axis represents the time of RFP expression in hours when the timing of chromosome segregation was set to zero for each cell. Y axis is the number of cells that expressed RFP at that time. Cell numbers plotted were n = 248 by DV and n = 346 by LSM880; total number, n = 594. These data are combined in this graph.

Fig 3: Effect of lamin depletion on localization of BAF and cGAS. (A and B) The localization and phosphorylation of BAF in WT, Lmna−/−, Lmnb1−/−, and Lmnb2−/− MEFs was analyzed by immunofluorescence (A) and immunoblotting, respectively (B). (A) Single confocal sections of the indicated cells stained with anti-BANF1/BAF (EPR7668), followed with Alexa Fluor 488-labeled anti-rabbit IgG, and Hoechst 33342 for DNA. (B) Whole cell lysates from the indicated cells were probed with anti-LA/C, anti-LB1/2, anti-GAPDH (as loading control), and anti-BANF1/BAF (EPR7668). (C and D) The localization and phosphorylation of BAF in WT MEFs expressing scrambled control, shLA, shLC or a combination of shLA and shLC was analyzed by immunofluorescence (C) and immunoblotting, respectively (D). (C) Single confocal sections of the indicated cells stained with anti-BANF1/BAF (EPR7668), followed with Cy5-labeled anti-rabbit IgG, and Hoechst 33342 for DNA. (D) Whole cell lysates from the indicated cells were probed with anti-LA/C, anti-GAPDH (as loading control), and anti-BANF1/BAF (EPR7668). (E and F) The localization and the expression levels of cGAS in WT, Lmna−/−, Lmnb1−/−, and Lmnb2−/− MEFs was analyzed by immunofluorescence (E) and immunoblotting, respectively (F). (E) Single confocal sections of the indicated cells stained with anti-cGAS, followed with Alexa 488-labeled anti-rabbit IgG, and Hoechst 33342 for DNA. (F) Whole cell lysates from the indicated cells were probed with anti-cGAS and anti-GAPDH (as loading control). (A–F) At least two independent experiments were performed. (A, C, and E) Bars: 20 μm. (B, D, and F) Positions of the size standards are shown on the right. The values on non-phospho-BAF, phospho-BAF and LA/C are densitometric quantitation normalized to GAPDH. Source data are available for this figure: SourceData F6.

Fig 4: FRAP of cytoplasmic and nuclear cGAS, and the accumulation kinetics of LC, BAF and cGAS at the rupture sites. (A–D) FRAP. A 2-µm diameter spot in a nucleus or cytoplasm was bleached, and the fluorescence recovery was measured for 30 s (A and B) and 360 s (C and D; means ± SEM; n = 20 and 17 cells for B and D, respectively, from two independent experiments). (A and C) The right four columns are magnified views of orange boxes, and the photobleaching sites are indicated with yellow dotted circles. Bars: 5 μm (the first column) and 2 μm (the second to fifth column). (E) Relative intensities of sfGFP-DARPin-LA6 (top), Halo-BAF (middle), and cGAS-sfCherry (bottom) at the rupture sites in cells with cytoplasmic or nuclear cGAS in Fig. 7, A–C (means ± SEM; n = 10 cells; *, P < 0.01; **, P < 0.001 by a linear mixed model).

Fig 5: The effect of re-expression of A-type lamins. (A) Validation of A-type lamins re-expression in Lmna-KO MEFs. Whole cell lysates from MEFs expressing the indicated constructs were probed with anti-LA/C and anti-GAPDH (as loading control). PB533-mEmerald-C1-Neo were used as control (empty). Positions of the size standards are shown on the right. The degradation fragment is indicated with #. The contribution of the fragment to rescue effects is likely to be marginal because the expression level is significantly low compared to mEmerald-LA (top band). (B–E) Representative images of single confocal sections of the indicated MEFs fixed within 10 min after laser microirradiation and stained with anti-BANF1 (3F10-4G12; B) or anti-cGAS (D), Cy5-labeled anti-mouse IgG or rabbit IgG, and Hoechst 33342 for DNA. The max intensities of BAF (C) and cGAS (E) signals at the rupture sites in the indicated MEFs. The plotted points are from two independent experiments (n = 30, 29, 32, and 32 for Empty, LA, LC, and LA + LC, respectively; significance was determined by a Kruskal–Wallis test, P < 0.05; far outliers were excluded if P < 0.001 by the Smirnov-Grubbs test; horizontal dotted lines show the mean values, *, P < 0.05 from empty control, †, P < 0.05 from LC by a Steel–Dwass multiple comparison after far outliers were excluded). (B and D) Bars: 20 μm. Yellow arrowheads indicate laser microirradiation-induced NE rupture sites. Blue arrowheads indicate spontaneously produced NE protrusions. Source data are available for this figure: SourceData F9.