Figure 4. Analysis of cell cycle, cell growth, and cell death following treatment of U87MG GBM with the ATMi inhibitor KU60019, CBD and γ-irradiation, alone or in combination.

(A and B) Changes in the cell cycle and levels of death were determined following treatments of U87MG cells with CBD (10-20 μM), ATMi (2 μM) and γ-irradiation at 10 Gy, alone or in combination. Cell cycle-apoptosis analysis was performed 30 h and 48 h after indicated treatments. CBD, ATMi or their combination, as well as control 0.1% DMSO, were added 30 min before irradiation. The nuclei of control and treated cells were stained with PI and DNA content in cells was determined using the flow cytometry. The nuclei of apoptotic and secondary necrotic cells were localized in the pre-G1 region. A typical result 30 h after treatment is shown in the panel (A). Pooled results of four independent experiments for U87MG 48 h after treatments are shown in panel (B). Error bars represent means ±S.D. (p < 0.05, Student's t-test). The stars, arrows, and circles indicate significant differences in the pre-G1 and the G2/M levels between cells after specified treatment. (C) Kinetics of U87MG cell growth was monitored 0-96 h after indicated treatments: 0.1% DMSO (as control), ATMi (2 μM), CBD (10 μM), γ-irradiation (at 5 Gy), alone or in combination. (D) Clonogenic survival assay was performed for cell cultures after indicated treatments with 0.1% DMSO (as control), ATMi (1-2 μM), CBD (20 μM) or a combination of ATMi+CBD without or with γ-irradiation at 5 Gy and 10 Gy. The final number of cell clones was determined after 12 days of cell culture growth. A ratio of a number of clones of treated cells to a number of clones of control cells reflects cell survival (percentage). Stars indicate the absence of survived clones.