| Autor: |
Theodore M. Kamenecka, Courtney A. Miller, Gavin Rumbaugh, Patrick R. Griffin, Thomas Vaissière, László Radnai, Rebecca F Stremel, William H. Martin |
| Rok vydání: |
2020 |
| Předmět: |
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| DOI: |
10.1101/2020.01.09.900365 |
| Popis: |
Cytokinesis is the last step of mitotic cell division that separates the cytoplasm of dividing cells. Small molecule inhibitors targeting either the elements of the regulatory pathways controlling cytokinesis, or the terminal effectors have been of interest as potential drug candidates for the treatment of various diseases. Here we have developed a cell-based assay for the discovery of novel cytokinesis inhibitors. The assay is performed in a 96-well plate format in 48 hours. Living cells, nuclei and nuclei of dead cells are revealed by a single staining step with three fluorescent dyes, followed by live cell imaging using an automated fluorescence microscope. The primary signal is the nuclei-to-cell ratio (NCR). In the presence of cytokinesis inhibitors, this ratio is expected to increase over time, as the ratio of multinucleated cells increases in the population. Cytotoxicity is quantified as the ratio of dead nuclei to total nuclei. A screening window coefficient (Z’) of 0.65 indicates that the assay is suitable for screening purposes, as the positive and negative controls are well-separated. EC50values for four compounds can be reliably determined in a single 96-well plate by using only six different compound concentrations. An excellent test-retest reliability (R2=0.998) was found for EC50values covering a ∼1500-fold range of potencies. The robustness, simplicity and flexibility of the assay is demonstrated here by using modulators of actin dynamics with different mechanisms of action (jasplakinolide, cytochalasin D and swinholide A) and derivatives of the nonmuscle myosin II inhibitor blebbistatin. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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