Cell Tracking.

A) Examples from Kasumi-1 cell and NB4 cell lines (upon the different conditions: DMSO, Palbociclib 50 nM, PF-0606873600 50 nM, Ribociclib 50 nM, Untreated), showing a tracked cell that explores the different cell-cycle phases (Scale bar is 5 µm). B) Fluorescence images with R and G channels were processed in Fiji to create a HSB stack, from which the Brightness and Hue channel were extracted to be used respectively as tracking channel and cell phase identification channel (Scale bar is 50 µm). C) Plot curves generated using the data extracted from the TrackMate script execution. The variations over time of mCherry (red curve) and mVenus (green curve) fluorescence intensities (left plot) and of the Hue scale (right plot) of a single NB4 cell in DMSO condition are shown.

Training and inference pipelines.

A) The Machine Learning pipeline followed to create the quality model. Using timetk time-series associated features are extracted from the list of manually annotated tracks. A random forest model is then trained to predict whether a track is cycling or not. B) An unannotated track can be fed to the model to predict whether it is cycling or not.

Cell Cycle Phase Assignment.

A) Waterfall plot of the “sorted” and “splitted” cells. Each row corresponds to a cell. B) Boxplot of the cell phase duration of the first cell cycle. The asterisks are the adjusted significance level given by Wilcox test of the sample for each over the NB4 DMSO for each phase according to the color (ns: p > 0.05, *: p <= 0.05, **: p <= 0.01, ***: p <= 0.001, ****: p <= 0.0001).

Experimental and analysis workflow.

Summary of the entire experimental workflow, from cell seeding to the calculation of cell cycle phases. Asterisks in the Live Cell Imaging section mean optional settings. Image created on Biorender.com.

© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

Cell Cycle Phase Assignment.

A) Examples from NB4 cell line upon different Palbociclib treatment concentrations showing a cell that explores the cell cycle phases (Scale bar is 5 µm). For Palbociclib 250 nM and Palbociclib 1250 nM conditions, the cell was manually tracked for presentation purposes.

A) Pearson correlation among pre and post time-lapse acquisition of Kasumi-1 (left) and NB4 (right) cell lines. In the x axis is reported the log1p counts mean between the two replicates before the acquisition, while on the y axis the log1p counts mean between the two replicates after the acquisition.

A) Boxplot showing the distribution of the feature extracted from the training set using timetk. B) ROC curve of the random forest model computed in the test set.

A) Waterfall plot of the “sorted” and “splitted” cells. Each row corresponds to a cell. B) Boxplot of the cell phase duration of the first cell cycle. A total of 1116 cells were analysed, obtaining a mean (± SD) cell cycle duration of 24.5 ± 8.5 h. C) Example of an adherent cell in control condition tracked with the described pipeline (Scale bar is 5 µm).