Radiations induce DNA damage, caspase-3 activation, and tumor repopulation in NSCLC cells. (A) Confocal images of immunostained A549 and H460 cells showing γH2AX foci following 8 Gy irradiation at 48 h. Scale bars: 25 μm. (B, C) The left panel shows flow cytometry analysis of A549 (B) and H460 (C) cell death after 0 Gy or 8 Gy irradiation on day 3. Apoptosis was monitored by Annexin V/propidium iodide (PI) double staining. The right panel shows quantitative analysis of early apoptosis and total cell death in 0 Gy- or 8 Gy-irradiated A549 (B) and H460 (C) cells (***p<0.001, Student’s t test, n = 3). (D) Cleaved caspase-3 induced by 8 Gy radiations was assayed by western blotting, and β-tubulin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as loading controls. (E) Representative confocal images of immunostained A549 and H460 cells showing cleaved caspase-3 following exposure to 8 Gy radiations on day 3. Scale bars: 25 μm. (F) The 8 Gy-irradiated NSCLC cells promoted the growth of living NSCLC reporter cells. The upper panel depicts luciferase activities showing the growth of A549 Fluc and H460 Fluc cells that were seeded alone or with 0 Gy- or 8 Gy-irradiated NSCLC cells. The lower panel shows the representative bioluminescence images (**p<0.01, ***p<0.001, one-way analysis of variance [ANOVA], n = 4).