Fig. 7. Protection of polyploid cells from apoptosis. After irradiation, cell samples were taken and assessed by two-channel FACS analysis for Annexin V and PI. Subsequently, cells were characterised according to their DNA content and the percentage of Annexin V-positive cells in each fraction compared. (A) The comparison of the proportion of Annexin V-positive Namalwa (A) and WI-L2-NS (B) cells in the 2C-4C (black squares) and >4C (black circles) ploidy fractions on days 2-4 after irradiation. For both Namalwa (P<0.01) and WI-L2-NS (P<0.02) cells, significant differences were detected (Student's t-test). Data represent mean values from three to five experiments±s.e.m. (C) In unirradiated control cells, 85% of Annexin V-positive cells showed PI fluorescence related to 2C-4C cells. (D) The proportion of Annexin V-positive cells was assessed on day 4 after 10 Gy in relation to the actual ploidy value. As can be seen, irradiation was gradually decreasing with each step of ploidy increase, suggesting the protection of endopolyploid cells or their positive selection by apoptosis, accompanying each round of DNA replication. (E) After irradiation, Namalwa cell samples were taken on day 3, stained and assessed by immunofluorescent microscopy for Annexin V (FITC, green) and Rad51 (Alexa Fluor 633; red). Annexin-V-positive apoptotic cells are Rad51-foci-negative and vice versa. DNA is counterstained with DAPI (blue). Scale bar: 40 µm.