Fig. 3.
Reducing LAP2β expression in lamin B1–deficient (B1KO) neurons with siLAP2β (a small-interfering RNA against Tmpo) increases NM ruptures and results in more cell death. (A) Transcript levels for Tmpo in Lmnb1+/+ (WT) and B1KO neurons that had been transfected with siLAP2β or a control siRNA (siControl). Bar depicts the mean in two experiments; dots show results in two independent experiments; transcript levels were normalized to Ppia transcripts. Neurons were examined 24 h after transfections. (B) Confocal micrographs of B1KO neurons transfected with siLAP2β or siControl. Neurons were stained with an antibody against LAP2β (white in the Left; red in the merged image). DNA was stained with DAPI (blue). (Scale bars, 10 μm.) (C) Percentages of siLAP2β- or siControl-transfected B1KO neurons with a NM rupture. ***P < 0.0005; χ2 test. Mean ± SEM; n = 3 independent experiments; 20 h of imaging/experiment. Dots depict percentages in three independent experiments. In three independent experiments, a total of 150 siLAP2β-transfected neurons and 125 control siRNA–transfected neurons were analyzed. (D) Ratio of the total number of NM ruptures to the number of neurons counted. Dots depict ratios in three independent experiments. *P < 0.05; unpaired Student’s t test. In three independent experiments, a total of 150 siLAP2β-transfected neurons and 125 control siRNA–transfected neurons were analyzed. (E and F) Bar graphs depict the mean time between NM ruptures (E) and the mean duration of NM ruptures (F) in siLAP2β- or siControl-transfected B1KO neurons (mean ± SD; >35 NM ruptures analyzed/group). ***P < 0.0005; unpaired Student’s t test. (G) Confocal micrographs of siLAP2β- or siControl-transfected B1KO neurons that expressed NLS-GFP (white). Larger numbers of NM ruptures (evident by escape of GFP into the cytoplasm and/or sudden loss of GFP fluorescence within the cell nucleus; yellow arrows) were observed in siLAP2β-transfected B1KO neurons. (Scale bars, 25 μm.) (H) Confocal micrographs of siLAP2β- or siControl-transfected B1KO neurons after staining with antibodies against LAP2β (red) and caspase 3 (white); DNA was stained with DAPI (blue). (Scale bars, 20 μm.)