Release from complete replication blockage by a high concentration of aphidicolin or HU induces DSBs.

<p>(A) Cell-cycle analysis after treatment with 2 mM HU for 2 h. The BrdU-positive fraction was quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060043#pone-0060043-g001" target="_blank">Figure 1</a>. (B) Frequency of chromosomal aberrations (CAs) for <i>wild-type</i> DT40 cells and <i>RAD54^−/−/KU70^−/−</i> cells. Cells were exposed to 2 mM HU for 2 h and then released in a drug-free medium for 3 or 6 h. (C) Cell-cycle analysis after treatment for 2 h with 0.5 µM aphidicolin (APH). The BrdU-positive fraction was quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060043#pone-0060043-g001" target="_blank">Figure 1</a>. (D) Frequency of chromosomal aberrations (CAs) for <i>wild-type</i> DT40 cells and <i>RAD54^−/−/KU70^−/−</i> cells. Cells were exposed with 0.5 µM aphidicolin (APH) for 2 h and released in a drug-free medium for 3 or 6 h. More than 50 cells were analyzed in each case. Error bars show standard error for the number of CAs in 50 mitotic cells, calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060043#pone-0060043-g002" target="_blank">Figure 2</a>. Asterisk and double asterisk: significant difference compared with <i>wild-type cells</i> (P<0.05).</p

Replication-blocking agents induce comparable numbers of chromosome breaks in both DSB-repair-proficient and -deficient chicken DT40 and human Nalm-6 cell lines.

<p>(A–D) Frequency of chromosomal aberrations (CAs) in <i>wild-type</i> and <i>RAD54^−/−/KU70^−/−</i> DT40 cells before (0) and after treatment with (A) γ-irradiation, (B) 5-FU, (C) HU, and (D) aphidicolin (APH). Cells were analyzed at 3 h after irradiation (A). Cells were incubated with 5-FU or HU for 24 h, or with aphidicolin for 48 h at the indicated concentrations (B–D). In each case, cells were treated with colcemid for the last 3 h. More than 100 cells were analyzed in each case. (E–H) Frequency of chromosomal aberrations (CAs) in <i>wild-type</i> and <i>RAD54^−/−/LIG4^−/−</i> Nalm-6 cells before (0) and after treatment with (E) γ-irradiation, (F) 5-FU, and (G) HU. Cells were incubated with 5-FU or HU for 48 h at the indicated concentrations (F, G). (H) The number of induced CAs was calculated by subtracting the number of non-treated cells from the number of cells treated with γ-rays or chemicals. More than 50 cells were analyzed at 8 h after irradiation at 0.1 Gy. More than 100 cells were analyzed for 5-FU and HU. Error bars show standard error, based on the Poisson distribution of spontaneous chromosomal aberrations observed previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060043#pone.0060043-Sonoda2" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060043#pone.0060043-Kikuchi1" target="_blank">[52]</a>.</p

Contribution of PIF1 and ATRIP to the prevention of chromosomal breakage.

<p>(A) Cells with the indicated genotype were exposed to the indicated replication-blocking agents and DNA damage agents. The dose of the agents is displayed on the x-axis on a linear scale, while the percent fraction of surviving cells is displayed on the y-axis on a logarithmic scale. Error bars show standard deviation of mean for three independent assays. (B) Frequency of chromosomal aberrations (CAs) in <i>wild-type</i>, <i>PIF1^−/−</i>, and <i>RAD54^−/−/KU70^−/−</i> DT40 cells before (0) and after treatment with aphidicolin at indicated concentration for 48 h. (C) Percentage of the cells carrying the indicated number of chromosomal breaks is indicated as a histogram. Indicated cells were treated with 0.1 µM aphidicolin (APH) for 24 h. More than 50 cells were analyzed in each case. Error bars show standard error for the number of CAs in 50 mitotic cells, calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060043#pone-0060043-g002" target="_blank">Figure 2</a>.</p

Comparable chromosomal breakage after removal of replication-blocking agents.

<p>Cells were exposed to 0.25 µM aphidicolin (APH) for 48 h. Frequency of chromosomal aberrations (CAs) for <i>wild-type</i> DT40 cells and <i>RAD54^−/−/KU70^−/−</i> cells is shown. More than 50 cells were analyzed in each case. Error bars show the standard error for the number of CAs in 50 mitotic cells, calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060043#pone-0060043-g002" target="_blank">Figure 2</a>. In each case, cells were incubated with colcemid for the last 3 h.</p

Phylogenomics Resolves The Timing And Pattern Of Insect Evolution: Supplementary File Archives.

Phylogenomics Resolves The Timing And Pattern Of Insect Evolution: Supplementary File Archives. This file includes 14 supplementary archives which are in detail described in the README