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

Figure 1.

Figure 1

Replication Forks Restart after Short Replication Blocks but Become Inactivated during Long Replication Blocks

(A) Labeling protocols for DNA fiber and replication foci analysis. U2OS cells were pulse-labeled with CldU, treated with 2 mM HU for the times indicated, and released into IdU. CldU was detected using a specific primary antibody and a secondary antibody in red. IdU was detected using specific primary antibody and a secondary antibody in green.

(B) Representative images of replication tracks from U2OS cells after release from 1, 2, or 24 hr HU treatment.

(C) Quantification of fork restart in U2OS cells after release from 1, 2, or 24 hr HU treatment.

(D) Global replication restart in U2OS cells after release from 2 or 24 hr HU treatment. Cells were pulse-labeled as in (A), then fixed and immunostained for CldU (red) and IdU (green). DNA was counterstained with DAPI (blue).

(E) Quantification of cells restarting global replication as in (D). Cells labeled with both CldU and IdU are shown as percentages of all cells labeled with CldU.

(F) Quantification of cells newly entering S phase after release from 2 or 24 hr HU treatment. Cells labeled with only IdU are shown as percentages of total cells. The means and standard deviation (SD) (bars) of at least three independent experiments are shown. Values marked with asterisks are significantly different (Student's t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; see also Figure S1).