Fig. 6.

Transduction of wild-type and TKO MEFs with a lentivirus encoding a LAP2β-tdTomato fusion protein prevents NM ruptures and reduces DNA damage. (A) Transcript levels for Tmpo (the gene for LAP2β) in nontransduced or LAP2β-tdTomato–transduced TKO MEFs (mean ± SEM; n = 3 independent experiments). Transcript levels were normalized to Ppia transcripts. **P = 0.0079; unpaired Student’s t test. (B) Percentages of nontransduced and LAP2β-tdTomato–transduced TKO MEFs that had a NM rupture event during 20 h of live-cell imaging. In 141 nontransduced TKO MEFs, 34 MEFs had a NM rupture. In 103 LAP2β-tdTomato–expressing MEFs, only 1 cell had a NM rupture. Mean ± SEM from three independent experiments (20 h of imaging/experiment). ***P < 0.0005; χ² test. Black dots depict percentages in each experiment. (C) Ratio of the number of NM ruptures in TKO MEFs and LAP2β-tdTomato–transduced MEFs over the total number of cells analyzed. In 141 nontransduced TKO MEFs, there were 69 NM ruptures. In 103 LAP2β-tdTomato–expressing MEFs, there was only one NM rupture. Mean ± SEM from three independent experiments; **P = 0.0055; unpaired Student’s t test. Black dots depict ratios in each experiment. (D) Percentages of nontransduced and LAP2β-tdTomato–transduced TKO MEFs with γH2AX foci. Mean ± SEM from three independent experiments; ***P < 0.0005; χ² test. Black dots depict ratios in each experiment. In three independent experiments, 50 nontransduced and 101 LAP2β-tdTomato–expressing MEFs were analyzed. (E) Percentages of nontransduced and LAP2β-tdTomato–transduced TKO MEFs with a NM rupture when plated on PDMS membranes under static conditions. In 172 nontransduced TKO MEFs, 29 MEFs had a NM rupture. In 195 LAP2β-tdTomato–expressing MEFs, there were no NM ruptures. Shown are mean percentages ± SEM from three independent experiments; ***P < 0.0001; χ² test. Black dots depict percentages in each experiment. (F) Percentages of nontransduced and LAP2β-tdTomato–transduced TKO MEFs with a NM rupture when the cells were subjected to uniaxial stretching on a PDMS membrane for 2 h. In 151 nontransduced TKO MEFs, 79 MEFs had a NM rupture. Among 198 LAP2β-tdTomato–expressing MEFs, three cells had a NM rupture. Shown are mean ± SEM from three independent experiments; ***P < 0.0001; χ² test. Black dots depict percentages in each experiment. (G) Immunofluorescence microscopy of nontransduced and LAP2β-tdTomato–transduced TKO MEFs under static and stretched conditions. The TKO MEFs expressed NLS-GFP (green) alone or NLS-GFP and LAP2β-tdTomato (red). DNA was stained with DAPI (blue). Magenta arrows point to NM ruptures. (Scale bars, 10 μm.)