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

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

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

Additional file 2: Table S2. of Temporo-spatial cell-cycle kinetics in HeLa cells irradiated by Ir-192 high dose-rate remote afterloading system (HDR-RALS)

Datasets for Fig. 7. (XLSX 10 kb

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

Additional file 4: Table S4. of Temporo-spatial cell-cycle kinetics in HeLa cells irradiated by Ir-192 high dose-rate remote afterloading system (HDR-RALS)

Datasets for Fig. 9. (XLSX 9 kb

Additional file 3: Table S3. of Temporo-spatial cell-cycle kinetics in HeLa cells irradiated by Ir-192 high dose-rate remote afterloading system (HDR-RALS)

Datasets for Fig. 8. (XLSX 9 kb

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