Figure 4.
Formation of 53BP1 foci in telomeric and non-telomeric DNA in GSCs, but not non-stem tumor cells. A, representative pictures of Hoechst dye staining to detect chromatin condensations in late apoptotic cells (indicated by arrows) treated with indicated dose of telomestatin (TMS) for 96 hours (left), and the graph indicating the proportion of apoptotic cells in 3 GBM spheres (right). *, statistical significance (P < 0.05). B, iFISH analysis indicating telomeric and non-telomeric DNA damages in telomestatin-treated GBM spheres. GBM146 spheres (GSCs) or serum-propagated GBM146 cells (non-GSCs) were treated with 1 µmol/L of telomestatin in serum-free medium for 96 hours and subjected to iFISH analysis (top). Red, telomeric DNA; green, 53BP1; blue, DAPI staining for nuclear DNA. Quantitative graph of the 53BP1 focus–positive cells (bottom). GBM146 cells were classified into the focus-positive or -negative fractions according to the number of cells with punctate nuclear 53BP1 foci (n > 2). *, statistical significance (P < 0.001). C, magnified views of the representative telomere dysfunction–induced foci (arrow in left) and non-telomeric DNA damage foci (arrow in right) in GSCs derived from GBM146 treated with 1 µmol/L of telomestatin for 96 hours. DAPI, 40′, 6-diamidino-2-phenylindole.