Research Paper Volume 15, Issue 2 pp 492—512

Figure 1. M3541 suppresses DNA DSB repair and modulates cellular response to radiation. (A). Dose-dependent inhibition of ATM autophosphorylation by M3541 in response to radiation. Proliferating A549, A375 and RKO cells were exposed to 5Gy IR to increasing concentrations of M3541 for 1 hour. Cell lysates were collected and ATM phosphorylation at serine 1981 was determined by MSD. Dashed line indicated 90% inhibition. (B) Analysis of γH2AX foci in A549 cells following exposure to DMSO, 1μM M3541, 5Gy IR, or combination of both for 0.5, 4 and 24 h by immunofluorescence. DAPI was used for nuclear counterstain and images were taken at 63x magnification. Scale bar is 10 μm. (C) Quantification of γH2AX foci was done by counting the number of cells with >10 foci in images from (C) after indicated times post radiation. (D) BrdU cell cycle analyses of A549 cells exposed to DMSO, M3541, IR or IR+M3541 for 24 h by flow cytometry. The percentage of cells in each phase was calculated and representative data are shown. (E) Western blot analysis of the effect of M3541 on IR-induced ATM and ATR signaling in A549 cell lysates prepared 6 h post IR. Representative images are shown.