(A) Left: Asynchronously cycling cells aligned by the end of the mitosis (anaphase), comparing CDK4/6, CDK2, and APC/CCdh1 activity changes. Corrected CDK4/6 activity signals are shown. Cells were classified by their CDK4/6 activity 2 hr after anaphase as being CDK4/6high or CDK4/6low, and a subset of CDK4/6low cells were further classified as CDK4/6delay if they had initially low CDK4/6 activity but later again increased CDK4/6 activity. Right: Activity histograms show that CDK4/6 but not CDK2 activity is rapidly activated with a bimodal activity distribution 2 hr after mitosis. Dotted line indicates the threshold used to classify the subpopulation of cells with CDK4/6, CDK2 or APC/CCdh1 activity on or off (n > 2,000 cells). (B) Examples of cells that either kept CDK4/6 activity on at mitotic exit (top), or exited to an approximately 2, 6 or 9 hr long transient G0 with low CDK4/6 activity (lower three panels). Corresponding CDK2 activities are shown to highlight that both CDK activities are low during a transient G0 and are sequentially activated (3 cells shown for each transient G0 time period). (C) A cumulative representation of the fraction of cells with active CDK4/6 (>0.7), hyperphosphorylated Rb (see Figure 2—figure supplement 1B), activated CDK2 (>0.76) and inactivated APC/CCdh1 as a function of time after mitosis (n > 2,000 cells). (D,E) Live-fixed analysis of cells in the same population with active or inactive CDK4/6 at different times after anaphase. The plot in (E) shows the fraction of cells that have Rb phosphorylated or not in cells were CDK4/6 activity is high or low as a function of time after anaphase (n > 2,000 cells).