Radiosensitivity in cells in early M phase is comparable to that in KPU-300–treated cells.

<p>(A) Fractions sorted by flow cytometry. Fr. 1, whole cell population; Fr. 2, cell fraction enriched in early M phase following the shake-off method; Fr. 3, cell fraction accumulated in early M phase following KPU-300 treatment for 24 h. (B) Radiosensitivity in each cell fraction following various treatments. Radiation dose was 4 Gy, and concentration of KPU-300 was 30 nM. A value of “-” in the “Fr.” row (i.e., lanes 1 and 4) indicates that cell sorting was not performed. The SF for Lane No. 9 was normalized by dividing the SF for Lane No. 8 by that for Lane No. 5. Data represent means ± S.E. of values obtained from three independent experiments. Error bars are not displayed when they would have been smaller than the circular symbol indicating the mean. <i>N</i>.<i>S</i>., not significant by either ANOVA or t-test.</p

Radiosensitivity and cell cycle kinetics of cells subjected to KPU-300 treatment after irradiation.

<p>(A) Survival curves in HeLa-Fucci cells treated with KPU-300 after irradiation. Cells were treated with 30 nM KPU-300 for 24 h immediately after irradiation, and then prepared for colony-forming assay. For normalization, the curve for combined treatment was shifted upward so as to obtain the surviving fraction 1 at 0 Gy. Data represent means ± S.E. of values obtained from three independent experiments. (B) Cell cycle kinetics after the same treatment described in Fig 7A. (a) Time course of DNA content with or without KPU-300 treatment after 2 Gy or 6 Gy irradiation. (b) Time course of two-dimensional flow-cytometric analysis to detect green fluorescence and an M-phase marker. The acquired time points are shown as hours:minutes in each image. (c) Quantitative analysis of green cells (left panel) and M-phase cells (right panel) after the same treatment described in Fig 7A. Data represent means ± S.E. of values obtained from three independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01 vs. lower values for the same time points.</p

Confocal fluorescence imaging of spheroids after treatment with KPU-300.

<p>The spheroid was treated with 30 nM KPU-300 and observed at the indicated times at the depth of 65 μm from the bottom using the confocal laser scanning fluorescence microscopy. The time points are shown as hours:minutes in each image. Bar, 200 μm.</p

Characterization of abnormal Fucci fluorescence following KPU-300 treatment.

<p>(A) Representative images of abnormal fluorescence after treatment with KPU-300. The time points are shown as hours:minutes in each image; 0:00 represents the start of drug treatment. Bar, 20 μm (B) Relationship between abnormal Fucci fluorescence and M phase following KPU-300 treatment. (a) Two-dimensional flow-cytometric analysis of Fucci fluorescence. The area within a quadrangle represents cells expressing abnormal Fucci fluorescence. (b) Two-dimensional flow-cytometric analysis of DNA content and phosphorylated histone H3 (pHH3). The area within a quadrangle represents cells in M phase. The acquired time points are shown as hours:minutes in each image; 0:00 represents the start of drug treatment. (c) Quantitative analysis of cells with abnormal Fucci expression and those in M phase in Fig 3a and 3b. Data represent means ± S.E. of values obtained from three independent experiments. *<i>p</i> < 0.05; **<i>p</i> < 0.01 vs. controls at time 0.</p

Characterization of cell cycle kinetics in HeLa-Fucci cells following KPU-300 treatment.

<p>(A) Chemical structure of KPU-300. (B) Immunostaining for β-tubulin. Exponentially growing HeLa-Fucci cells were fixed and prepared for immunostaining following treatment with 30 nM KPU-300 for 16 h. Blue, DAPI; pink, β-tubulin. Bar, 5 μm. (C) Time course of Fucci fluorescence and histogram of DNA content following KPU-300 treatment. Cells were treated with the indicated concentrations of KPU-300 and prepared for time-lapse imaging and flow-cytometric analysis. The time points are shown as hours:minutes in each image; 0:00 represents the start of drug treatment. Bar, 20 μm. (D) Time course of the percentages of green fluorescent cells (a), M-phase cells (b), and total cell number (c) following KPU-300 treatment. Green cells and M-phase cells were manually counted in merged fluorescence and phase contrast images. A total of 170–350 cells obtained from 8–11 visual fields were counted in one experiment. M phase cells adopt a round-shape, accompanied by disappearance of the nuclear envelope. Data represent means ± S.E. of values from three independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01 vs. treatment with 10 nM for the same duration of time.</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

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

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

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