PMC

The canonical Rb/E2F network is highlighted with a dashed rectangle. CycD and CycE represent Cyclin D/CDK4/6 complex and Cyclin E/CDK2 complex, respectively.

(a) Scatter plot of commitment as determined by Amp under different c-Myc inhibitor (10058-F4) concentrations (80, 90 and 100 μM). 101, 100 and 93 cells were analysed for each condition. (b) Histogram of cell division as determined by Amp under the conditions in a. (c) Dose–response curves (fitted to data points as Hill functions) indicate the proportion of divided/committed cells under different c-Myc inhibitor concentrations. (d) Representative E2F1 dynamics trajectories (green for committed cells; red for uncommitted cells) at 90 μM 10058-F4 inhibitor concentration.

(a) hE2F1p::4NLS-d4Venus reporter construct. Box with cross indicates that 5′ long terminal repeat (LTR) is inactivated after viral integration. (b) Time-lapse microscopy images of REF52 rat fibroblasts expressing hE2F1p::4NLS-d4Venus reporter released from serum starvation by adding 10% bovine growth serum (BGS). Upper panel, phase channel; low panel, Venus channel. Scale bar, 50 μm. (c) E2F1 dynamics trajectories of the five individual cells shown in b. Time-series raw data (sampled per hour) were smoothened in a 3-h time window. (d) Characterization of E2F1 dynamics trajectory in divided (green) cells with defined metrics: t₁, initial delay; t₂, activation time; t₃, post-activation time; Amp, amplitude; S (shadow area), total E2F1 work during activation time; k, slope; T, the entire cell cycle length. Orange triangle indicates E2F1 signal upturn time point and the blue one indicates cell division time point. (e) Histogram shows the distribution of Amp at different serum levels. (f–h) Histograms show distributions of t₁, t₂ and t₃ (in divided cells) at different serum levels. The mean values were compared among different serum concentrations and plotted as insets.

(a–c) Normalized Amp (to the Amp for base parameter values) within 120 h under the inhibition of CDK4/6, CDK2 or Myc as function of inhibition rate. Horizontal dashed lines mark that the expected threshold value for normalized Amp_th is 0.5, given that the measured Amp_th is around half of the average Amp of cells (2.5 versus 5, ). (d) Log sensitivity analysis of Amp to inhibition rate. (e–g) Normalized t₂ to its initial level as a function of inhibition rate under each inhibition case. Yellow shading highlights the region where cells are expected to commit into cell cycle. (h) Log sensitivity analysis of t₂ to inhibition rate. For k_InhM, log sensitivity was calculated within the committed region. (i) The central motif of Myc/Rb/E2F network includes E2F auto-regulation (module for level control) and its titration by Rb (module for timing control). (j) Steady-state analysis of the central motif under the inhibition of Rb phosphorylation through reduction of cyclin complexes activity. The decrease in Rb phosphorylation rate leads to only a slight reduction (ΔAmp) in steady-state E2F level. Black and grey lines are E2F synthesis curves under low (black) and high (grey) cellular unphosphorylated Rb levels, respectively. Red line indicates the degradation curve. Blue line indicates the threshold Amp_th. Green dots, stable steady states; red dots, unstable steady states. (k) Steady-state analysis of the central motif. Black and grey lines are E2F synthesis curves given high (black) and low (grey) synthesis rate, respectively.

(a) Linking E2F dynamics to phenotypes in single cells. (b) The experimental design to correlate E2F dynamics with commitment into cell cycle entry. Serum-starved cells were released by adding 1% BGS together with EdU and then incubated under time-lapse microscope for E2F1 dynamics measurement. After 44 h incubation, cells were fixed and subjected to EdU staining. (c) EdU labelling after E2F dynamics measurement and cell fixation. Circles mark the nuclei and white arrows indicate cells without EdU signal. Scale bar, 50 μm. (d) Scatter plot based on cell fate (circle, uncommitted; dot, committed) as determined by Amp. Dotted line indicates Amp_th (at which an individual cell has 50% probability to commit into cell cycle). It was determined by using data analysis in f. Solid line indicates the boundary of signal between EdU-positive and -negative cells. 113 cells were analysed. (e) Histogram based on cell fate (red, undivided; green divided) as determined by Amp. 254 cells were analysed by pooling data points from 10, 2 and 1% BGS experimental conditions. (f) Logistic regression curves indicate the probability of cell commitment (dashed line) or division (solid line) as predicted by Amp. The shaded area around each logistic regression curve indicates a 95% confidence interval for 1,000 bootstrapping iterations.

(a) Scatter plot based on commitment as determined by Amp under inhibition of CDKs (PD0332991 for CDK4/6 and CVT-313 for CDK2). Committed cells were measured by EdU labelling after 48 h incubation, and divided cells were counted 48 h after addition of 10% BGS. (b) Histogram based on cell division as determined by Amp under inhibition of CDKs (48 h observation). (c) Proportion of committed/divided cells under different inhibitions of CDKs during a 48- and 92-h (the latter only for the combined inhibition) observation window. The exact number of total cells followed and that of cells committed/divided are indicated on top of each error bar. (d) Four representative E2F1 dynamics trajectories under the same conditions as a. Each colour bar on top indicates the length of t₂ of its corresponding trajectory. (e) Distribution of t₂ of individual cells in mock, CDK4/6-, CDK2- and combinational inhibited samples. Mean±s.d. is shown on top right of each panel. 54 (Vehicle), 106 (15 nM PD0332991), 102 (100 nM PD0332991), 109 (1 μM PD0332991), 94 (1 μM CVT313), 105 (5 μM CVT313) and 101 (1 μM PD0332991 and 5 μM CVT313) cells were analysed for each condition, respectively. (f) Plot of the first cell cycle length (T) under the condition as in e. Every dot represents a single cell. Black cross indicates the median of T among the cells divided within 48 h. *P=0.01855, Wilcoxon one-side test. For all testing results, P<0.005 unless marked.