Fig 1: DMAPT reduces NHEJ activity in NSCLC cells by decreasing chromatin binding of key NHEJ proteins and inhibiting auto-phosphorylation of the DNA–PK complexa NHEJ measured by pEJ GFP reporter flow cytometry assay in DMAPT-treated cells. A549 pEJ cells were treated with DMAPT for 24 h following transfection of an I-SceI plasmid. The percentages of GFP-positive cells were analyzed by flow cytometry. Values were compared to non-transfected control. Bars show mean ± SD (n = 3). b Western immunoblotting of nuclear and chromatin fractions of Ku80 and XRCC4 in A549 cells following DMAPT treatment and/or IR. Cells were harvested 24 h following DMAPT treatment and 2 h following IR treatment. c Western immunoblot showing DNA-PK-Ser2056 phosphorylation levels following DMAPT treatment and/or 10 Gy IR in A549 cells. Cells were harvested 24 h following DMAPT treatment and 1 h following IR treatment. d Representative immunofluorescence images of DNA-PK-pS2056 focus formation following DMAPT and/or IR treatment in A549 cells. Cells were fixed 24 h following DMAPT treatment and 1 h following IR treatment. Results in NCI-H460 and NCI-H1299 cells are shown in Supplementary Fig. S6. Scale bar = 10 µm. e Quantification of 53BP1 foci in A549 cells treated with DMAPT and/or 10 Gy IR over a 24-h period. Bars show mean ± SD (n = 3). f Representative images of e. Scale bar = 10 µm. g DMAPT-induced changes in higher-order chromatin structure were assayed by chromatin digestion with increasing amounts of MNase followed by agarose gel electrophoresis. *indicates p < 0.05 by t-test