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. 2022 Feb 9;135(3):jcs258831. doi: 10.1242/jcs.258831

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© 2022. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 3. — PLK1 imparts survival against PolST-mediated cell death. (A) Crystal Violet staining showing enhanced survival of the PLK1-overexpressing stable cell line in U2OS background in the presence of PolST expression. (B) The survival exhibited by PLK1 to PolST-induced cell death was reversed in the presence of PLK1 inhibitor BI-2536 (50 nM). Cells were stained after 1 week. (C) Timeline showing the experimental strategy for live-cell time-lapse imaging for cells in which PolST was induced for 48 h (PolST was induced for 32 h before imaging and the cells were then monitored for 16 h). (D) Representative still images from live-cell time-lapse imaging of PLK1-overexpressing U2OS cells in the presence of PolST (48 h induction), showing some cells dividing successfully in comparison to the controls (arrows indicate reference cells). (E) Graphical representation of the live-cell imaging analysis: PLK1-overexpressing U2OS cells in the presence of PolST (48 h induction) showed a lower percentage of apoptotic cells in comparison to the control cells (the percentage of cells not undergoing mitosis is not shown). Cells in prometaphase-like stage include the cells that were delayed in prometaphase for at least 800 min until the end of experiment. (Time taken from NEBD to anaphase onset was considered to calculate the duration of mitosis. NEBD was observed by chromosomal condensation and appearance of irregular nuclei, whereas anaphase onset was observed by separation of the two chromosome masses.) (F) Dot plot analysis after live-cell imaging of PolST cells in U2OS background (in the presence and absence of doxycycline for 48 h), showing the time taken for cells in mitosis. Twenty mitotic cells were included from each group for the analysis, and the cells undergoing death are not represented in the analysis. The cells that were delayed in a prometaphase-like stage for a minimum of 800 min until the end of the experiment have been categorized separately.