Figure 2. AZD6738 promotes G2-M transition after irradiation and increases residual DNA damage.
(A) Top: Representative cell cycle distributions in NCI-H460 pretreated with AZD6738 for 6 hours before irradiation and analyzed 6 hours after radiation. Bottom: Representative mitotic populations determined by staining for histone H3 phosphorylated at serine 10 (H3S10ph). Inset squares illustrate H3S10ph-positive cells. (B and C) Percentages of cells in G2 were calculated with FlowJo v10.7 (BD Life Sciences) in (B) NCI-H460 cells and (C) NCI-H1299 cells. (D and E) Numbers of mitotic cells were calculated using FlowJo in (D) NCI-H460 cells and (E) NCI-H1299 cells. (F) Representative immunofluorescence images of foci in NCI-H1299 cells treated with AZD6738 1 hour before irradiation and analyzed for γH2AX and 53BP1 foci as a surrogate for DNA damage 24 hours after irradiation. Original magnification, ×20. (G) γH2AX and (H) 53BP1 foci in H1299 cells exposed to photons or protons. At least 10,000 cells were analyzed per group, and error bars represent the SD. Statistical significance was assessed with 2-way ANOVA with Tukey’s multiple-comparison test. NS, non-significant. *P < 0.05; ***P < 0.001; ****P < 0.0001.