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. 2021 Jun 24;12:3937. doi: 10.1038/s41467-021-24224-3

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a DNA fiber analysis of the replication fork speed and symmetry in nontreated conditions. Left: schematic of the CldU/IdU pulse-labeling protocol used. Middle: analysis of the replication fork speed. Total number of molecules analyzed: siCTRL (n = 541), siTPR (n = 567). The plot includes the mean ± SD. Statistical test: unpaired t-test with Welch’s correction, two-tailed. Right: analysis of the replication fork symmetry. The ratio between CldU/IdU was calculated from values in a (left). Whiskers indicate the 5th–95th percentiles (p value associated with Kolmogorov–Smirnov test). b DNA fiber analysis of the ability of forks to stop progression in the presence of replication stress. Top: schematic of the CldU/IdU pulse-labeling protocol used. Bottom: percentage of the double-labeled replication forks containing both CldU and IdU signal is showed. Total number of slides analyzed from three independent experiments: siCTRL (n = 6), siTPR (n = 9). The plot includes the mean ± SD. Statistical test: unpaired t-test with Welch’s correction, two-tailed. c DNA fiber analysis of the replication fork recovery upon HU removal. Left: schematic of the CldU/IdU pulse-labeling protocol used. Middle: length of the IdU tracks measured upon hydroxyurea removal. Total number of molecules analyzed: siCTRL (n = 297), siTPR (n = 310). The plot includes the mean ± SD. Statistical test: unpaired t-test with Welch’s correction, two-tailed. Right: analysis of the replication fork symmetry. The ratio between CldU/IdU was calculated from values in c (middle). Whiskers indicate the 5th–95th percentiles. Statistical test: unpaired t-test with Welch’s correction, two-tailed. d Categories of DNA replication intermediates identified in TEM analysis with a schematic representation of the molecule with dsDNA in black and ssDNA in red. Scale bars of 360 nm, which correspond to 1 kb of DNA, are reported on each picture. e Chart reporting the percentages of RIs identified. Gapped forks are defined as forks with ssDNA gaps at fork junction points longer than 200 nts. Data are represented as mean ± SD from four independent experiments (n = 4) for hydroxyurea-treated (HU) samples and two independent experiments (n = 2) for nontreated samples. Statistical analysis: two-way ordinary ANOVA followed by Sidak’s multiple comparison test.