Extended Data Fig. 3: Cyclin E induces CDK2 and E2F activation in CDK4/6 inhibited cells.
From: An intermediate Rb–E2F activity state safeguards proliferation commitment
Related to Fig. 2. a, Single-cell traces of CDK2 activity, E2F activity, and CRL4Cdt2 activity reporter intensity after release with growth media + DMSO for 48 h (n = 25 cells each. 1 of n = 3 biological replicates). b, Cumulative frequency of CDK2-activated, E2F-activated, and S phase-entered cells after release with growth media + DMSO. A time gap between E2F-active and S phase entry was calculated at the 50% line (n = 1941 cells. 1 of n = 3 biological replicates). c, d, Left, E2F activity traces in cells treated with DMSO and CDK2i (1 μM) 14 h after release with starvation media + EGF (20 ng/mL). Right, Representative CRL4Cdt2 reporter traces in cells treated with DMSO 14 h after release with starvation media + EGF (20 ng/mL) to show examples of computational gating. Cells that were in G1 phase (c) or S phase (d) upon the drug treatment were computationally gated based on the CRL4Cdt2 reporter signal. p-values for E2F activity 6 h after the drug treatment (20 h after release) were calculated using two-sided, two-sample t tests (E2F in c: mean ± SE. n = 465, 454 cells for DMSO, CDK2i, respectively. p-value = 9.2 × 10−10. E2F in d: mean ± SE. n = 66, 88 cells for DMSO, CDK2i, respectively. p-value = 0.014. CRL4Cdt2: single-cell traces. n = 15 cells each. 1 of n = 3 biological replicates). e, f, CDK2 (left) and E2F (right) activity traces in control or Dox-inducible HA-tagged cyclin E1-expressing cells. Dox was added 5 h before release to induce cyclin E1. Cells were released with growth media + CDK4/6i (1 μM) (e: single-cell traces. n = 5 cells each. f: mean ± SE. n = 205, 363 cells for Con, Cyclin E1, respectively. 1 of n = 3 biological replicates). g, Same data in f was gated for cells entered S phase and plotted as a phase-plane trajectory. Intervals between arrows are one hour. Blue circles mark the time points for S phase entry, which were determined by the CRL4Cdt2 reporter signal (n = 6, 175 cells for Con, Cyclin E1, respectively. 1 of n = 3 biological replicates). h, Left, percentage of cells entered S phase by 40 h after release in control or Dox-inducible HA-tagged cyclin E1-expressing cells. Dox was added 5 h before release to induce cyclin E1. Cells were released with growth media + CDK4/6i (1 μM). A p-value was calculated using two-sided, two-sample t tests (mean ± SE. n = 3 biological replicates. n = 703, 834 cells in total for Con, Cyclin E1, respectively. p-value = 7.2 × 10−5). Right, histograms of cyclin E1-HA intensity in control or Dox-inducible HA-tagged cyclin E1-expressing cells. Cells were fixed 48 h after release with growth media + CDK4/6i (1 μM) to stain HA and confirm cyclin E1 induction. (n = 1420, 708 cells for Con, Cyclin E1, respectively. 1 of n = 3 biological replicates). i, Same data in Fig. 2g (S enter) was shown with CDK2 activity traces. Cells were released with starvation media + EGF (20 ng/mL) + CDK4/6i (1 μM). Dashed lines mark the time points for S phase entry, which were determined by the CRL4Cdt2 reporter signal (n = 3 cells. 1 of n = 3 biological replicates). j, Same data in Fig. 2c was shown as representative single-cell traces. CDK2 (top) and E2F (bottom) activity traces after release with growth media + CDK4/6i (1 μM). Cells were stratified and color-coded based on the time cells spend from E2F-active to S phase entry. Cell traces were computationally aligned at S phase entry (n = 3 cells each. 1 of n = 3 biological replicates). k, Box plots of CDC6 mRNA puncta area multiplied by its intensity. Cells were starved for two days for measuring CDC6 mRNA levels in G0. Cells were fixed 36 h after release with growth media + CDK4/6i (1 μM) for measuring CDC6 mRNA levels in G1 and S. G1 and S cells were gated based on the CRL4Cdt2 reporter signal. Box centers are median values, box edges are the 25th and 75th percentiles, and whiskers are minimum and maximum values. A p-value was calculated using two-sided, two-sample t tests (n = 1410, 1124, 102 cells for G0, G1, S cells, respectively. p-value (G0 vs G1) = 2.5 × 10−298. 1 of n = 3 biological replicates).
