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Open Access Highly Accessed Methodology article

Drug-induced cell cycle modulation leading to cell-cycle arrest, nuclear mis-segregation, or endoreplication

Asako Sakaue-Sawano12, Tamiyo Kobayashi3, Kenji Ohtawa4 and Atsushi Miyawaki12*

Author Affiliations

1 Life Function and Dynamics, ERATO, JST, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan

2 Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan

3 MIS Division, Olympus Corp., 2-3 Kuboyama-cho, Hachioji, Tokyo 192-8512, Japan

4 Brain Science Research Division, Brain Science and Life Technology, Research Foundation, 1-28-12 Narimasu, Itabashi, Tokyo 175-0094, Japan

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BMC Cell Biology 2011, 12:2  doi:10.1186/1471-2121-12-2

Published: 13 January 2011

Additional files

Additional file 1:

Construction of Fucci2. (A)Constructs with concatenated mCherry and mVenus fused to deletion mutants of human Cdt1 and Geminin. cyan box, Cy motif; pink box, D (destruction) box; black box, NLS (nuclear localization signal). (B) Excitation (broken line) and emission (solid line) spectra of mVenus and mCherry. (C) Fucci2 labels individual G1 phase nuclei in red and S/G2/M phase nuclei yellowish green. (D,E) Characterization of HeLa/Fucci2 cells. Cells showing red [mCherry(+)mVenus(-)], orange [mCherry(+)mVenus(+)], and yellowish green [mCherry(-)mVenus(+)] fluorescence were collected (D), and their DNA contents were stained with Hoechst33342 and measured using FACS (E).

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Additional file 2:

Time-lapse imaging of Fucci2-expressing HeLa cells. HeLa/Fucci2 cells were grown on a glass-bottom dish and time-lapse imaging was performed using an LCV100 microscope (Olympus). Images were acquired every 15.5 minutes (m). Playback speed is 14,000 × real time. Total imaging time = 96 hours (h).

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Additional file 3:

Time-lapse imaging of Fucci2-expressing NMuMG cells. NMuMG/Fucci2 cells were grown on a glass-bottom dish and time-lapse imaging was performed using an LCV100. Images were acquired every 22.5 m. Playback speed is 7,200 × real time. Total imaging time = 50 h.

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Additional file 4:

Time-lapse imaging of Fucci2-expressing HeLa cells during etoposide treatment. HeLa/Fucci2 cells were grown on a glass-bottom dish and time-lapse imaging was performed using an LCV100. Cells were treated with 1 μM etoposide. Cells at 4 different positions were imaged every 20.5 m. Playback speed is 7,000 × real time. Total imaging time = 49 h. Most cells were arrested at the G2 DNA checkpoint (yellowish green).

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Additional file 5:

Time-lapse imaging of Fucci2-expressing NMuMG cells during 1 μM etoposide treatment. NMuMG/Fucci2 cells were grown on a glass-bottom dish and time-lapse imaging was performed using an LCV100. Cells were treated with 1 μM etoposide. Images were acquired every 22.5 m. Playback speed is 7,200 × real time. Total imaging time = 50 h. Most cells were entered M phase and then underwent "nuclear mis-segregation."

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Additional file 6:

Time-lapse imaging of Fucci2-expressing NMuMG cells during 10 μM etoposide Treatment. NMuMG/Fucci2 cells were grown on a glass-bottom dish and time-lapse imaging was performed using an LCV100. Cells were treated with 10 μM etoposide. Images were acquired every 22.5 m. Playback speed is 7,200 × real time. Total imaging time = 50 h. The yellowish green-to-red color conversions are indicated by white arrows. Most cells were showed transition from the mitotic to endoreplication cycle.

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Additional file 7:

Summary of NMuMG/Fucci2 cell responses to etoposide. Overview diagram of cell populations of normal cycling or G2 arrest, nuclear mis-segregation characterized by chromosome fragmentation, tetraploid cells generated by endoreplication, and cell death. Typical fluorescence images of the first three patterns are shown (inset). Scale bars, 10 μm.

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Additional file 8:

An NMuMG/Fucci2 Cell Approaching 8C. In the presence of 3 μM etoposide, a cell with a yellowish green nucleus in the normal cycling stayed at G2 checkpoint for some time. Then the cell underwent endoreplication to have a red nucleus, which was further converted to a yellowish green one. The yellowish green-to-red color conversion is indicated by an white arrow. No vestiges of mitosis were observed. Scale bar, 10 μm.

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Additional file 9:

Time-lapse imaging of Fucci2-expressing NMuMG cells during 3 μM etoposide treatment (Additional file 8). NMuMG/Fucci2 cells were grown on a glass-bottom dish and time-lapse imaging was performed using an LCV100. Cells were treated with 3 μM etoposide. Images were acquired every 22.5 m. Playback speed is 15,000 × real time. Total imaging time = 50 h. Only a small fraction of the NMuMG cells were observed to go though multiple endoreplication cycles like the cell featured in this movie.

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Additional file 10:

An NMuMG/Fucci2 Cell Reaching 8C. In the presence of 1 μM etoposide, a cell with a yellowish green nucleus underwent endoreplication until 8C DNA content. The yellowish green-to-red color conversions are indicated by white arrows. No vestiges of mitosis were observed. Scale bar, 10 μm.

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Additional file 11:

Time-lapse imaging of Fucci2-expressing NMuMG cells during 1 μM etoposide treatment (Additional file 10). NMuMG/Fucci2 cells were grown on a glass-bottom dish and time-lapse imaging was performed using an LCV100. Cells were treated with 1 μM etoposide. Images were acquired every 27 m. Playback speed is 16,700 × real time. Total imaging time = 88 h. Only a small fraction of the NMuMG cells were observed to go though multiple endoreplication cycles like the cell featured in this movie.

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