Figure S3. Cyclin A2-eYFP cytoplasmic appearance after modulation of cyclin dependent kinase (CDK) activity.

(A) Quantification of nuclear (left) and cytoplasmic (right) accumulation of CycA2-eYFP in at least 17 single RPE-CycA2-eYFP cells treated with DMSO (top), RO-3306 (middle), or Nu6140 (bottom) for 4 h; each track represents a single cell. Cells were selected based on clear but low cytoplasmic CycA2-eYFP signal at time point of inhibitor addition, and synchronised in silico at the time point when cells reached a threshold of cytoplasmic CycA2-eYFP (indicated with the grey dotted line). Average intensities (below). Error bars show SD. Please note that differences after 2 h involve that DMSO-treated cells enter mitosis, giving an increase in average fluorescence upon cell rounding followed by a drop in fluorescence upon mitotic CycA2-eYFP degradation. (B) Quantification of at least 95 single RPE-CycA2-eYFP cells upon treatment with either DMSO (control) or Wee1 inhibitor (MK-1775). Cells were treated with the drugs and tracked using live-cell imaging. Each track represents one single cell, the black dot represents the first time point when cytoplasmic CycA2 can be detected and the red dot indicates mitotic entry. Overlap of the cumulative appearance of cytoplasmic CycA2 in the two different treatments (right). (C) Western blot of indicated cell lines transfected with CDK1, CDK2, or scrambled (Control) siRNAs for 48 h. Samples were prepared from 4 wells to 96-well plate to mimic conditions used for microscopy. (D) Proportion of RPE cells passing through mitosis during 16 h after knockdown of CDK1 or CDK2 for 48 h (left). Duration of mitosis in the indicated knockdown conditions (right).

Source data are available for this figure.