Fig. 4. Confocal microscope analysis of cultured HeLa cells during Fas-mediated apoptosis. Cells grown on glass coverslips were untreated (A-E) or treated with Fas antibody and PD 98059 for 24 hours (F-T) and prepared for immunofluorescence microscopy. Samples were double-stained with NuMA (A, F and K) and lamin B (B, G and L) or lamin A/C (P) and lamin B (Q) antibodies. FITC-conjugated rabbit-anti-mouse (A, F, K and P) and TRITC-conjugated rabbit-anti-goat (B, G, L and Q) secondary antibodies were used. Samples were counterstained for DNA with Hoechst (D, I, N and S; Hoechst stain is shown in glow color). In the beginning of apoptosis NuMA condenses in the center of the nucleus (F) and excludes the condensed chromatin in the nuclear periphery (arrows). Lamin B shows a folded staining pattern (G). Note that lamin B staining shows the upper surface of the nuclear lamina in a normal interphase cell. At the end of apoptosis, NuMA partially encircles the NuMA-negative nuclear fragments (arrows) within apoptotic bodies (K). Lamin B remains around NuMA and the fragmented nuclei (L). Lamin A/C colocalizes with lamin B (P-R). Transmission views show typical apoptotic features including shrinking of the cell and nucleus, cytoplasmic blebbing, detaching of the cells and finally formation of the apoptotic bodies (E, J, O and T). Bar 10 µm.