Skip to main content
. 2010 Feb 26;37(4):492–502. doi: 10.1016/j.molcel.2010.01.021

Figure 2.

Figure 2

Replication Forks Accumulate DNA Damage during Long Replication Blocks

(A) Colocalization of HU-induced γH2AX with RPA. Cells were left untreated or treated with 2 mM HU for 2 or 24 hr, fixed, and immunostained for γH2AX (green) and RPA70 (red). DNA was counterstained with DAPI (blue).

(B) Quantification of U2OS cells displaying γH2AX immunostaining after 0, 1, 2, or 24 hr of treatment with 2 mM HU and 1 hr after release from 2 or 24 hr HU treatment. Cells containing more than 10 foci were scored as positive. The means and SD (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (∗∗p < 0.01).

(C) Pulsed-field gel electrophoresis to visualize DSB induction in U2OS cells treated with 2 mM HU for 2 to 48 hr.

(D) Colocalization of γH2AX and stalled replication forks in U2OS cells. Cells were pulse-labeled with CldU for 20 min, treated with 2 mM HU for 2 or 24 hr, and released into IdU for 1 hr. Cells were immunostained for CldU (red), IdU (green), and γH2AX (far red), and DNA was counterstained with DAPI (blue). DNA was denatured with HCl to allow CldU/IdU detection.