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Fig. 2. Changes in lamina morphology during FKC8-induced apoptosis. (A) Human MSCs at passage four were transduced with lentivirus vectors encoding lamin A-DsRed, histone H4-CFP or lamin B-GFP. Cells were finally transduced with FKC8. Control cells were left without AP20187 treatment (mock-treated), and FKC8 was activated by incubating the cells with 100 nM AP20187 for 6 hours. Confocal images were taken from representative nuclei. Images were processed with TeloView and show views of the xy-axis (square) and xz-axis (rectangle) of cells. Bar, 5 µm. (B) Visualization of fragmented DNA and the lamina after FKC8 activation. Cells expressing FKC8 were treated with 100 nM AP20187 for 6 hours or were left without AP20187 treatment. Fragmented DNA (green) was visualized with the Cell Death Detection kit, whereas the lamina was detected with anti lamin B1 antibody (red). Short arrows indicate convoluted nuclei and long arrows indicate nuclei with degraded lamin B. Bar, 10 µm. (C) The effect of Z-VAD.fmk treatment on lamina organization. Human MSCs at passage four were transduced with lamin B-GFP and FKC8 lentiviral vectors. Cells were treated with 50 nM Z-VAD.fmk for two days and 10 nM AP20187 was added during the last 5 hours (+AP+Z). Control cells were treated with 10 nM AP20187 only (+AP). Confocal images were taken from living cells. Images obtained after TeloView processing show maximum projections in xy-axis and xz-axis in green, and a single optical section in the xy-axis in gray. Bar, 5 µm. (D) Statistical analyses of changes in nuclear depth upon caspase-8 activation. Human MSCs at passage four were subsequently transduced with vectors coding for lamin B-GFP and CC8 or FKC8. Control cells were transduced with lamin B-GFP vector only. CC8-transduced cells were examined 16 hours post-transduction. FKC8-transduced cells were treated with either 10 nM or 100 nM AP20187 for 5 hours. Nuclear depth was measured from confocal images. 120 nuclei were evaluated.
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