Wound healing assay is a simple and cost-effective in vitro assay for assessing therapeutic impacts on cell migration. its key limitation is the possible confoundment by other cellular phenotypes, causing misinterpretation of the experimental outcome. in this study, we attempted to address this problem by developing a simple analytical approach for scoring therapeutic influences on both cell migration and cell death, while normalizing the influence of cell growth using Mitomycin C pre-treatment. By carefully mapping the relationship between cell death and wound closure rate, contribution of cell death and cell migration on the observed wound closure delay can be quantitatively separated at all drug dosing. We showed that both intrinsic cell motility difference and extrinsic factors such as cell seeding density can significantly affect final interpretation of therapeutic impacts on cellular phenotypes. Such discrepancy can be rectified by using the actual wound closure time of each treatment condition for the calculation of phenotypic scores. finally, we demonstrated a screen for strong pharmaceutical inhibitors of cell migration in cholangiocarcinoma cell lines. our approach enables accurate scoring of both migrastatic and cytotoxic effects, and can be easily implemented for high-throughput drug screening.

Forkhead box protein M1 (FOXM1) is a proliferation-associated transcription factor contributing to the G2/M phase transition of the cell cycle. Although the upregulation of FOXM1 has been observed in different cancer types, how the regulation of FOXM1 dynamically alters during cell cycles and potentially contributes to tumorigenesis is not well understood. We showed here the development and application of a tunable FOXM1-DHFR (FOXM1-D) sensor that enables surveillance and manipulation of the FOXM1 abundance. Using trimethoprim (TMP) to stabilize the sensor, we measured the kinetics of FOXM1-D production, degradation, and cytosolic-to-nuclear translocation in the G1 and G2 cell-cycle phases. By controlling FOXM1-D stability in different synchronized cell cycle pools, we found that the G1- and S-synchronized cells finished their first cell division faster, although the G2-synchronized cells were unaffected. Our analysis of single-cell FOXM1-D dynamics revealed that the two-round dividing cells had a lower amplitude and later peak time than those arrested in the first cell division. Destabilizing FOXM1-D in the single-round dividing cells enabled these cells to re-enter the second cell division, proving that overproduction of FOXM1 causes cell cycle arrest and prevents unscheduled proliferation.

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