Fig. 5. Subcellular distribution of proteins form the nuclear envelope during apoptosis. Monolayer cell cultures were incubated in the absence, -STSP (a-r) or presence, +STSP (a’-r’) of 1 µM staurosporine. After 15 hours of treatment the cells were fixed and permeabilized and subjected to double immunolabeling using the antibodies indicated. The chromatin was stained using Hoechst 33258. In stably transfected BHK cells, POM121-GFP was visualized by its own fluorescence (g,g’). The smaller panels show fields of cells. The larger panels show enlargements of control or stage II apoptotic nuclei inside the frames. Pairs of images of control and treated cells were acquired and processed identically. Control cells displayed an intense staining around the nuclear rim with all antibodies tested and nuclei with dispersed chromatin and large diameters (a-r). Apoptotic cells (a’-r’) showed nuclei with condensed chromatin and a smaller nuclear diameter. The immunolabeling of the periphery of apoptotic nuclei was weaker or absent. However, the immunofluorescence using mAb414 antibodies was retained (b’,j’,m’) in most apoptotic cells, although it frequently displayed a clustered distribution (j’). Bar, 20 µm.
