Figure 1.

Induction of DSBs by NCS. (A) Micrographs of live HeLa cell nuclei expressing both H2B-GFP and 53BP1-mCherry are shown under the following conditions: control (under physiological conditions) and upon addition of 200, 500, and 800 ng/mL NCS. The first column shows the H2B-GFP signal, the second column the 53BP1-mCherry signal with the nuclear contour (white line), and the third column the overlay of H2B-GFP (green) and 53BP1-mCherry (red) signals. (B) A nucleus treated with 500 ng/mL NCS (top row) and a control nucleus (bottom row), both formaldehyde-fixed, are shown. H2B-GFP (green) signal visualizes chromatin. 53BP1-mCherry (red) and γH2AX (blue) signals visualize the DNA repair foci and DSBs, respectively. The overlay of the red and blue signals shows their colocalization (pink) in the NCS-treated nucleus, whereas no DNA damage is found in the control nucleus. (C) An enlarged view of the boxed-in region from (B) with yellow circles highlighting the position of 53BP1 foci is shown. (D) Distribution of γH2AX signal over the population of 53BP1 foci (N = 1987 over 36 cells) is shown. The histogram shows the presence of three distinct Gaussian peaks (red, pink, and orange dashed lines), with means at discrete values, where the means of the second and third peaks are about twofold and threefold the values of the first peak’s mean, respectively. This suggests that the first peak indicates the fraction of 53BP1 foci containing a single DSB, the second peak two DSBs, and the third peak three DSBs. Scale bars, (A)–(B) 5 μm, (C) 1 μm. To see this figure in color, go online.