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Fig. 5. Mutations in lamin A or lamin B genes that make them caspase-3-sensitive affect lamina and telomere spatial organization. (A) Western blot analysis of lamin degradation in 293T cells. 293T cells were transduced with lentiviral vectors expressing wild-type lamin B-GFP, lamin B-L158D-GFP, wild-type lamin A-DsRed or lamin A V221D-GFP. Subsequently, half of the transformed cells were transduced with LV-CC8 (+). Proteins were extracted 18 hours after transduction and subjected to Western blot analysis. Lamin B-GFP was detected with an anti-GFP antibody and lamin A-DsRed was detected with an anti-lamin A/C antibody. Long arrows indicate the fused protein and small arrows the cleaved products. (B) Quantification of lamina morphologies in hMSCs. Human MSCs at passage four were transduced with lentivirus vectors coding for wild-type lamin B-GFP, lamin B L158D-GFP, wild type lamin A-DsRed, or the lamin A V221D-DsRed. Lamina morphology was determined by fluorescence microscopy. Histograms show the percentage of cells exhibiting intranuclear structures (purple) or degraded (red) lamina structures (as shown in Fig. 2). Maximum projections of confocal images show telomere organization in cells expressing lamin mutants. (C) hMSCs at PS6 were transduced with Trf1-DsRed (red) and lamin B L158D-GFP (green). In cells expressing lamin A V221D-DsRed (red), TRF2 was detected by immunostaining (blue). After TeloView processing, maximum projections in the xy-axis and xz-axis (in green), and a single section in the xy-axis (gray) are shown. Bar, 5 µm.
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