Fig. 3: Protein and phosphorylation dynamics during mitotic exit.

A Experimental workflow of cell synchronisation coupled to MS analysis. G2 phase cells pre-synchronised in G1 with palbociclib (Palbo) were incubated with DMA for 4 h. Prometaphase-arrested cells were harvested by mitotic shake-off and released in fresh medium. 5 h after release, early G1 cells were collected. For MS analysis, cells were lysed and protein digested with trypsin, followed by TMT labelling and phosphopeptides enrichment. B Simplified data analysis workflow used to define proteins and phosphorylation events significantly changing in prometaphase arrest and early G1. C Volcano plot showing limma adjusted p-values versus absolute limma log₂ fold change of protein abundance in prometaphase arrest and early G1. Statistically significant proteins are shown in blue, non-statistically significant proteins are shown in grey. Dotted lines represent cut-offs used to define oscillating proteins (limma adjusted p ≤ 0.01, log₂ fold change ≥ |0.5|). CCNB1 is significantly enriched in prometaphase arrest. D Volcano plot showing limma adjusted p-values versus absolute limma log₂ fold change of phosphorylation sites abundance in prometaphase arrest and early G1. Statistically significant phosphorylation sites are shown in red, non-statistically significant phosphorylation sites are shown in grey. Dotted lines represent cut-offs used to define oscillating phosphorylation events (limma adjusted p ≤ 0.01, log₂ fold change ≥ 1). Phosphorylation at Ser670 of BUBR1 is significantly enriched in prometaphase arrest. E Fisher’s exact test Gene Ontology (GO) analysis of proteins significantly changing in the Mitotic Exit dataset. The top three significantly enriched terms for each category (Reactome, Biological Processes, Molecular Function and Cellular Components) are shown. F Heatmap showing oscillating proteins detected in the Time Course dataset clustered based on the abundance profile of known cell cycle markers using T-distributed stochastic neighbour embedding. Protein changes are coloured according to their abundance (min-max normalised values). Bar plot on the right shows curve fold change (oscillation score) in grey and proteins significantly differentially expressed in prometaphase or early G1 in the Mitotic Exit dataset in blue. Clusters are grouped by colour and their respective reference protein is reported on the right. G Scatterplot comparing protein changes in the Mitotic Exit (fold change) and Time Course datasets (curve fold change). Statistically significant proteins are shown in blue, non-statistically significant proteins are shown in grey. Point size denotes −log10 q-values from the Time Course dataset. Representative cell cycle markers are labelled and proteins from the Cell Cycle-Dependent (CCD) set are coloured blue. H Overview of the datasets presented in this study. The Time Course dataset covers seven cell cycle stages from late G1 to M/G1. The Mitotic Exit dataset involves prometaphase arrest and early G1. The Cell Cycle-Dependent (CCD) set is defined as the union of proteins and phosphorylation sites that exhibit changes in the Time Course and Mitotic Exit datasets. The total number of proteins and phosphorylation sites in each dataset is shown.