Fig. 3

a, b Representative examples of double immunolabeling for γH2AX and 53BP1 in dissociated cortical neurons from non-irradiated (a) and irradiated rats (15d post-IR) (b). Some neurons exhibited a PDDF immunolabeled for γH2AX and 53BP1. Scale bar: 5 μm. c Western blot analysis of γH2AX in parietal cortex lysates from non-irradiated and irradiated rats (n = 3 animals per group). Protein levels of γH2AX were increased upon DNA-damage induced IR. The expression of histone H3 was used as protein loading control, and the fold increase estimated. d Proportion of cortical neurons containing γH2AX-positive PDDF in non-irradiated and irradiated neurons. (***p < 0.001 by Student’s t test). e Mean number of PDDF per nucleus within the PDDF-containing neuronal population. (*p < 0.05 by Student’s t test). f Distribution of PDDF in three nuclear regions: perinucleolar, nuclear periphery and nuclear interior. Approximately 70% of PDDF were spatially associated with the nucleolus in both non-irradiated and irradiated cortical neurons. g-l Double labeling for γH2AX in combination with 53BP1 (g, h), UBF (j), B23 (k) or histone H4K20me3 (l), and for 53BP1 in combination with WRAP53 (i) illustrating the concentration of γH2AX, 53BP1 and WRAP53 in PDDF, and the spatial association of PDDF with the nucleolus (j, k) and with heterochromatin masses (l). g: non-irradiated neuron. h-l: irradiated neurons at 15 days post-IR. Scale bar: 5 μm