Figure 7.

Blebbishield-derived cells (BD Cells) are susceptible to Hoechst-33342 and are heterogeneous in molecular profile. (a) Low Hoechst-33342 positivity and vacuolation in colonies derived from blebbishields. Blebbishields were allowed to differentiate in normal MEM and the colonies were stained with live stain Hoechst-33342 and imaged. Note the nuclei stained with low intensity similar to that of side-population cells (arrowheads). RT4P blebbishield-derived cells had at least one highly vacuolated cell per colony (hollow arrow). Note the DNA content in vacuoles of RT4P blebbishield-derived cells (solid arrows). The interior of the colony displayed no intense Hoechst-33342 positivity even after 90 min of incubation in Hoechst-33342 containing medium. (b) Effect of Hoechst-33342 in sphere survival. Hoechst-33342 (10 nM) was treated during or after blebbishield generation (24 h) and the surviving spheres were plotted. (c) Heterogeneity analysis of colonies (C1–C5) derived from single blebbishields of RT4P and RT4v6 cells by western blotting. Note the difference in levels of expression of myc isoforms, cleaved granzyme-B, s-FasL, active caspase-8, active caspase-3, cleaved p70S6K and cleaved XIAP. Asterisks, bands with major differences between colonies