a, Control and additional experiments to Fig. 3d. CDK2 activity traces of cells exposed to a control pulse or a 20-min NCS pulse (10 ng ml−1, marked in purple), followed by a 5-h incubation in different concentrations of mitogens (marked in grey). After the 5-h incubation, cells were replenished with full growth media. Cells undergoing mitosis within 1 h of replacement with full growth media were selected. b, Individual traces of CDK2 activity showing the time window when mitogens were withdrawn or MEK inhibited (100 nM PD0325901) for 5 h until cells were fixed (marked in grey). Cells that were fixed during the first 2 h after mitosis were selected. c, Percentage of hyper-Rb (Ser807/S811) in response to mitogen withdrawal and MEK inhibition. Data are mean ± s.d. (n = 2 biological replicates). d, Representative images of mRNA and protein levels of cyclin D1 (CCND1) and p21 (CDKN1A) after a 5-h period of mitogen withdrawal or MEK inhibition. Scale bar, 50 μm. e, mRNA and nuclear protein levels of p21 in response to mitogen withdrawal and MEK inhibition. Data are mean ± s.d. (n = 2 biological replicates). f, Expression level of nuclear cyclin D1 protein, normalized by initial level (time 0 h), in response to the translation inhibitor, cycloheximide (10 μg ml−1) (n = 2 biological replicates). g, Cyclin D1 protein (left) and mRNA (CCND1; right) levels, normalized by initial level (time 0 h), after mitogen withdrawal. The half-life of CCND1 mRNA was measured with or without the transcription inhibitor actinomycin D (ActD; 5 μg ml−1) (n = 2 biological replicates).