Fig. 2. Cell-type-specific modulation of NHEJ repair by cAMP signaling. (A, B, C) Effects of cAMP signaling upon the disappearance of γ-H2AX formed by γ-ray irradiation in SK-MEL-28 cells (A), HeLa cells (B), and A549 cells (C). The cells were irradiated with γ-ray, then incubated for 10 min before PGE2 treatment and collected at the presented time points for analysis by western blotting. The empty circles present vehicle-stimulated control cells, and the filled circles present PGE2- stimulated cells. (D) cAMP signaling effects upon NHEJ repair efficiency. The cells were cotransfected with GαsQL and SceI-linearized GFP fluorescent reporter plasmids using Lipofectamine 3000 and harvested after 24 hours for flow cytometric analysis. The NHEJ repair efficiencies were determined as the ratios of green fluorescence emitted from the repaired reporter plasmid to the red fluorescence emitted from the DsRed control plasmid. (E, F, G) PGE2 effects upon the recruitment of XRCC4 and DNA-ligase IV after exposure to γ-rays of SK-MEL-28 cells (E), HeLa cells (F), and A549 cells (G). The empty bars present XRCC4, and the filled bars present DNA-ligase IV. After pretreatment with PGE2, the cells were irradiated with γ-rays. The irradiated cells were reacted with antibodies against XRCC4 or DNA-ligase IV, followed by DAPI staining. The number of γ-H2AX foci per cell was derived by analyzing the confocal images of stained cells. The means ± standard error calculated from the three independent experiments were presented as columns. The asterisk (*) denotes statistically significant differences compared with the respective control (P ≤ 0.05, Mann-Whitney U test).