期刊
JOURNAL OF CELL SCIENCE
卷 130, 期 2, 页码 512-520出版社
COMPANY OF BIOLOGISTS LTD
DOI: 10.1242/jcs.195164
关键词
Automated microscopy; DNA replication origin; Drug synergy; FUCCI; Live-cell imaging; Taxane
类别
资金
- Cancer Research UK [C14303/A17197]
- Pancreatic Cancer UK Future Research Leaders fund
- Cancer Research UK [15678] Funding Source: researchfish
- Pancreatic Cancer UK [FLF2015_03_Cambridge] Funding Source: researchfish
The fluorescence ubiquitination-based cell cycle indicator (FUCCI) is a powerful tool for use in live cells but current FUCCI-based assays have limited throughput in terms of image processing and quantification. Here, we developed a lentiviral system that rapidly introduced FUCCI transgenes into cells by using an all-in-one expression cassette, FastFUCCI. The approach alleviated the need for sequential transduction and characterisation, improving labelling efficiency. We coupled the system to an automated imaging workflow capable of handling large datasets. The integrated assay enabled analyses of single-cell readouts at high spatiotemporal resolution. With the assay, we captured in detail the cell cycle alterations induced by antimitotic agents. We found that treated cells accumulated at G2 or M phase but eventually advanced through mitosis into the next interphase, where the majority of cell death occurred, irrespective of the preceding mitotic phenotype. Some cells appeared viable after mitotic slippage, and a fraction of them subsequently re-entered S phase. Accordingly, we found evidence that targeting the DNA replication origin activity sensitised cells to paclitaxel. In summary, we demonstrate the utility of the FastFUCCI assay for quantifying spatiotemporal dynamics and identify its potential in preclinical drug development.
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