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Fig. 4. Spatial organization of telomeres during FKC8-induced apoptosis in hMSCs. (A) Human MSCs at passage four were transduced with lentivirus vectors encoding either Trf2-citrine (shown in green) or Trf1-DsRed (shown in red), followed by a transduction with the lamin A-DsRed (red) or the lamin B-GFP (green) encoding vectors. The cells were finally transduced with FKC8. Confocal images were taken from living cells before (mock-treated) or 4-6 hours after 100 nM AP20187 treatment (+AP20187). Images were processed with TeloView to quantify the fluorescence dots. Images show representative nuclei. Bar, 5 µm. (B) Quantification of telomere fluorescence intensity. The fluorescent dots obtained in A were sorted and plotted according to their intensity. The intensity graph obtained from the control cells is shown in yellow, and that obtained from FKC8-activated cells, showing a convoluted nuclear shape, is shown in green. (C) Statistical analyses of telomere organization. Images of mock-treated or AP20187-treated cells, as described in A, were processed in TeloView. Statistical analyses of Trf1-DsRed spatial organization included the percentage of telomeres in close spatial distance, the percentage of fluorescent particles in aggregates, and the ratio between the two intensity slopes. AP20187-treated cells (+AP) were sorted according to the lamina shape. Forty nuclei were evaluated. (D) DIPimage image analysis shows one optical section along the xz-axis of lamin B-GFP-expressing (green) and Trf1-DsRed-expressing (red) cells. Images of representative nuclei within the subclasses, yellow, red, blue and green of C (a, b, c and d, respectively) were processed in DIPimage. Yellow shows overlap between lamin B-GFP and Trf1-DsRed (indicated by arrows). Bar, 5 µm.
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