Imaging studies revealed the activation of molecules involved in DNA sensing pathways in response to 6 Gy irradiation of B16F10 tumor cells. (a) The accumulation of micronuclei in cell cytosol after irradiation; scale bar = 20 μm (Ctrl, IR), 5 μm (inset); n = 13–15. (b) Irradiation-dependent accumulation of dsDNA in the cytosol of cells. Arrows are pointed to representative dsDNA spots; scale bar = 20 μm (Ctrl), 30 μm (IR), 15 μm (inset); n = 12–16. (c) Irradiation induced translocation of STING from cytoplasm to peri-nuclear punctate structures (arrows); scale bar = 30 μm (Ctrl, IR), 15 μm (inset); n = 5. (d) Irradiation induced translocation of IRF3 transcription factor from the cytoplasm to the nucleus; scale bar = 30 μm (Ctrl, IR), 15 μm (inset); n = 5. (e) Irradiation failed to induce a significant translocation of IRF7 transcription factor from the cytoplasm to the nucleus; scale bar = 20 μm (Ctrl, IR), 12 μm (inset); n = 9. (f) Irradiation induced translocation of NF-κB transcription factor from the cytoplasm to the nucleus; scale bar = 30 μm (Ctrl, IR), 18 μm (inset); n = 12 cells. Statistical significance was determined by one-way ANOVA followed by a Dunnett’s multiple comparisons test. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. Ctrl group; n = number of fields of view. Images of control nonirradiated cells (denoted by Ctrl), and irradiated cells 48 h after 6 Gy irradiation (denoted by IR) in all imaged channels are presented. In the graphs, x axis label Ctrl represents quantified values of control (nonirradiated) cells and labels IR 24 h and IR 48 h represent quantified values of 6 Gy irradiated cells 24 and 48 h after irradiation, respectively. ns = not significant.