Fig. 4.

Higher levels of LAP2β expression in differentiated B1KO neurons prevents NM ruptures. WT and B1KO neurons were transduced with a lentivirus encoding a LAP2β-tdTomato fusion protein. (A) Fluorescence intensity in nontransduced and LAP2β-tdTomato–transduced B1KO neurons. Mean ± SD ***P < 0.0001; unpaired Student’s t test. After staining neurons with an antibody against LAP2β, confocal micrographs were recorded and 10 images were analyzed by ImageJ. Fluorescence intensity for each nucleus was quantified and normalized to nuclear area (in square millimeters). (B) Immunofluorescence microscopy of nontransduced and LAP2β-tdTomato–transduced B1KO neurons. Cells were stained with an antibody against LAP2β (white on the Left; red in the merged image). DNA was stained with DAPI (blue). (Scale bars, 10 μm.) (C) Time-lapse microscopy study of NLS-GFP–expressing B1KO neurons (green) that had been incubated with a LAP2β-tdTomato lentivirus. The neurons were not subjected to drug selection; hence, some of the neurons did not express LAP2β-tdTomato (red). (Top) Micrographs of a field containing NLS-GFP–expressing B1KO neurons that were successfully transduced with LAP2β-tdTomato. NM ruptures were never observed in NLS-GFP–expressing B1KO neurons that expressed LAP2β-tdTomato. (Bottom) Confocal micrographs of another field in which several cells expressed NLS-GFP but no LAP2β-tdTomato. Neurons that expressed only NLS-GFP exhibited NM ruptures (evident by escape of GFP into the cytoplasm) (magenta arrows). (Scale bars, 20 μm.) (D) Percentages of NLS-GFP–expressing nontransduced B1KO neurons (n = 148) and LAP2β-tdTomato–transduced B1KO neurons (n = 101) that had at least one NM rupture (detected by escape of GFP into the cytoplasm). Mean ± SEM; n = 3 independent experiments; 20 h of imaging/experiment. Dots depict percentages in three independent experiments. ***P < 0.0005; χ² test. No NM ruptures were detected in 101 LAP2β-tdTomato–transduced B1KO neurons. A total of 148 nontransduced B1KO neurons were evaluated in the three experiments. (E) Ratio of the total number of NM ruptures in nontransduced and LAP2β-tdTomato–transduced B1KO neurons divided by the total number of neurons analyzed. Mean ± SEM; n = 3 independent experiments; ***P < 0.0005; unpaired Student’s t test. No NM ruptures were observed in LAP2β-tdTomato–transduced B1KO neurons. A total of 148 nontransduced B1KO neurons were evaluated. (F) Percentages of nontransduced and LAP2β-tdTomato–transduced B1KO neurons with γH2AX foci. A total of 240 LAP2β-tdTomato–transduced B1KO neurons and 156 nontransduced B1KO neurons were analyzed. Mean ± SEM; n = 3 independent experiments. ***P < 0.0005; χ² test. The total number of neurons scored are recorded with each bar. (G) Confocal micrographs of nontransduced and LAP2β-tdTomato–transduced B1KO neurons after staining the cells with antibodies against LAP2β (red) and the DNA damage marker γH2AX (white). (Scale bars, 10 μm.)