Dose-dependency of prolongation of red and green phase after irradiation.

<p>(A) Schematic presentation of irradiation timing and measurement of the duration of green (a) or red (b) phase. Black square represents M phase. In panel b, the two daughter cells did not exhibit exactly the same red-phase duration; therefore, each value was separately measured and mean values were calculated. (B) Dose-dependency of prolongation of red and green phases after irradiation. Data represent means ± S.D. of values obtained from at least four different fields for each dose. Each field contained at least 10 cells. *, p < 0.05; **, p < 0.01 vs. control values at 0 Gy (one way ANOVA with post hoc Dunnett’s test).</p

Characteristics of green-phase elongation in cells irradiated in G1 phase.

<p>(A) Pedigree analysis in cells irradiated during G1 phase. Rearrangement of cell population irradiated in G1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128090#pone.0128090.g003" target="_blank">Fig 3D</a>, according to the length of red phase (a). Rearrangement of panel a after subtraction of G1-phase duration (b). (B) Quantitative relationship between stage within G1 phase (cell No.) at the time of irradiation and durations of green phase, derived from Fig 4A. Regression line: y = 0.0197x + 11.447, r = 0.372, p < 0.001. (C) Comparison of green-phase duration between cells irradiated in early (red phase duration > 4 h) and late (red phase duration ≤ 4 h) G1 phases, using data from Fig 4A. Data are represented as box-and-whisker plots, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128090#pone.0128090.g003" target="_blank">Fig 3E</a>. For both populations, n = 100. **, p < 0.01 by Mann–Whitney U test. (D) Comparison of red-phase durations between cells with shorter (≤ 13 h) and longer (> 13 h) green-phase durations after irradiation in G1 phase, using data from Fig 4A. Data are represented as box-and-whisker plots, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128090#pone.0128090.g003" target="_blank">Fig 3E</a>. For both populations, n = 100. **, p < 0.01 by Mann–Whitney U test.</p

Cell-cycle progression is disrupted after irradiation.

<p>(A) A red cell enters M phase faster than a green cell after irradiation. Cell #1, a green cell; Cell #2, a red cell. Round cells represent M phase. Time is shown as hours:minutes. (B) Distribution of red- and green-phase durations in cells irradiated in red or green phase. Black and orange represent M phase and early S phase, respectively. The dotted line indicates the time when the first cell irradiated in red phase reached M phase. (C) Flow-cytometric detection of EdU-labeled mitotic cells after irradiation. Experiments were performed as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128090#pone.0128090.g001" target="_blank">Fig 1E</a>, except that cells were flash-labeled with EdU immediately after 5 Gy irradiation. (D) Rearrangement of sub-populations in Fig 3B. RPS: G1 population from which the G1-phase duration (red portion) was subtracted; GP1, S/G2 population from the left side of the dotted line; GP2, S/G2 population from the right side of the dotted line. (E) Duration of green phase after irradiation in each sub-population shown in Fig 3D. Data are represented as box-and-whisker plots showing the full range, 25–75% interquartile range (box), and median (bar) from 200 cells in RPS, 138 cells in GP1, and 289 cells in GP2. **, p < 0.01 by Mann–Whitney U test.</p

Estimation of G2 arrest durations in cells irradiated in G1, early/mid/late S, and G2 phases.

<p>(A) Pedigree analysis of cells irradiated in green phase. a: Distribution of total green-phase durations in cells irradiated in each phase, sorted according to green fluorescence intensities. Two straight lines represent the remaining S-phase durations at irradiation (maximal 7 h for the leftmost cell in early S phase) (lower dashed line) and plus elongation of S phase (maximal 2 h for the leftmost cell in early S phase) (upper dashed line) in each phase. b: Distribution of G2-arrest durations after subtraction of the corresponding S phase and its elongation from the left panel a. (B) Comparisons of G2-arrest durations in cells irradiated in each phase. Data are represented as box-and-whisker plots as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128090#pone.0128090.g003" target="_blank">Fig 3E</a>. Cell number in each sub-phase is equivalent to that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128090#pone.0128090.g005" target="_blank">Fig 5C</a>. *, p < 0.05; **, p < 0.01 by Mann–Whitney U test.</p

Cell-cycle kinetics in p53-functional cells following irradiation.

<p>(A) Order of progression to M phase in red and green cells in non-irradiated BJ1-hTERT-Fucci cells. Mitotic, red, and green cells at the start of observation were monitored until the next entry into M phase. (B) Pedigree analysis on Fucci fluorescence in irradiated BJ1-hTERT-Fucci cells. Time-lapse imaging was performed following 5 Gy irradiation. Each color represents the same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128090#pone.0128090.g001" target="_blank">Fig 1A and 1D</a> except that the white box represents early G1 phase without any fluorescence after mitotic skipping.</p

Durations (h) of each cell-cycle phase in exponentially growing HeLa-Fucci cells.

<p>1) From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128090#pone.0128090.g001" target="_blank">Fig 1A</a>, G1-phase duration was determined from the red-phase duration. S+G2-phase duration was determined from the green-phase duration. Values represent means from 150 cells. Mitotic cells were identified morphologically. Total cell-cycle time was calculated from the duration between one M phase and the next M phase, as determined by time-lapse imaging.</p><p>2) Each cell-cycle phase duration was calculated from the proportion of each cell cycle phase (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128090#pone.0128090.g001" target="_blank">Fig 1B</a>), assuming an exponential distribution according to the equations as described by Watanabe and Okada [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128090#pone.0128090.ref029" target="_blank">29</a>]. Values in parenthesis were derived from Fucci analysis.</p><p>3) Durations of S and G2 phases were directly determined from the curve in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128090#pone.0128090.g001" target="_blank">Fig 1C</a>, as described in Materials and Methods. G1-phase duration was determined by subtracting the S+G2+M duration from the total cell-cycle time.</p><p>4) The actual culture doubling time obtained from growth curves was 17–18 h.</p><p>Durations (h) of each cell-cycle phase in exponentially growing HeLa-Fucci cells.</p

Establishment of a method for classifying green phase into early/mid/late S and G2 phases.

<p>(A) Experimental procedure for classification of green phase. Time-lapse imaging was performed for cells flash-labeled with EdU immediately after 5 Gy irradiation, followed by EdU staining. Time is shown as hours:minutes. (B) Classification of green-phase cells into early/mid/late S and G2 phases, according to the method described in A. For simplicity, a subset of cells in each phase is presented. To adjust the fluorescence intensities among different fields, background fluorescence intensity of each image was normalized to 1. (C) Comparison of green fluorescence intensities among sub-groups. Data are represented as box-and-whisker plots, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128090#pone.0128090.g003" target="_blank">Fig 3E</a>, from 60 cells in early S, 139 cells in mid S, 101 cells in late S, and 127 cells in G2 phase. *, p < 0.05; **, p < 0.01 by Mann–Whitney U test. (D) Validation of the methodology, using GP1 and GP2 from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128090#pone.0128090.g003" target="_blank">Fig 3D</a>. Cell numbers in each sub-phase within GP1 (a) and GP2 (b) were determined. GP1 and GP2 consisted of 138 and 289 cells, respectively.</p

Endoreduplication rarely occurs in HeLa-Fucci cells following 5 Gy irradiation.

<p>(A) DNA-content analysis by flow cytometry. Cells were fixed at the indicated times after 5 Gy irradiation, and DNA content was quantitated by flow cytometry. Percentages of polyploid cells are indicated. (B) Detection of normal mitosis and mis-segregation following 5 Gy irradiation. a: normal mitosis. Yellow and white arrowheads represent two examples of normal mitosis. b: mis-segregation. Yellow arrowhead represents an example of mis-segregation. Of more than 600 cells analyzed, only a few cells exhibiting mis-segregation were detected under these conditions. Time is shown as hours:minutes.</p

Characterization of cell-cycle kinetics in exponentially growing HeLa-Fucci cells.

<p>(A) Pedigree analysis from one M phase to the next M phase. Real-time imaging analysis was performed based on 150 HeLa-Fucci cells. Each bar represents one cell cycle, starting from one M phase to the next M phase, consisting of M (black), red, orange, and green phases. Orange represents cells in which both red and green fluorescence signals were emitted during early S phase. (B) DNA-content analysis by flow cytometry using Hoechst 33342. Proportion of each cell-cycle phase is indicated. (C) Percent-labeled mitosis index. Cells were flash-labeled with EdU, and labeled mitotic cells were detected by flow cytometry. Two-dimensional analysis (phospho-histone H3 and EdU) was performed at the indicated times after flash-EdU labeling. (D) Order of progression to M phase in red and green cells. Mitotic, red, and green cells at the start of observation were monitored until the next entry into M phase. (E) Time course of EdU-labeled mitotic cells. Representative two-dimensional flow-cytometric plots are shown at the indicated times after flash EdU-labeling described in Fig 1C.</p