Fig. 1: A marked variation in the activation kinetics of E2F revealed by live cell E2F transcription analysis.
From: An intermediate Rb–E2F activity state safeguards proliferation commitment
a, Model of the cell cycle decision process in G1 phase. b, Schematic of the E2F transcriptional reporter. c, Experimental design for mitogen release in MCF-10A cells. d, Top, time-course images of a cell expressing mVenus (E2F reporter) and H2B–iRFP (nucleus marker), starved for 2 days and released with growth medium. Arrows indicate cell nuclei. Scale bar, 20 μm. Bottom, mVenus intensity (in arbitrary units (a.u.)) in the cell shown in the top (1 out of 3 biological replicates). e, mVenus intensity traces in control (Con) or doxycycline-inducible HA-tagged cyclin D1-expressing cells. Doxycycline was added 5 h before release to induce cyclin D1. Cells were released with starvation medium + EGF (20 ng ml–1). Cyclin D1-expressing cells were released with DMSO or CDK4/6 inhibitor (CDK4/6i; 1 μM palbociclib). HA > 212 was used to gate cyclin D1-overexpressing cells. P = 2.8 × 10−92 (mVenus intensity 16 h after release), calculated using two-sided, two-sample t-tests (mean ± s.e.). n = 500, 500 and 456 cells for Con + DMSO, cyclin D1 + DMSO and cyclin D1 + CDK4/6i, respectively; 1 out of 3 biological replicates. f, mVenus intensity traces after release with growth medium. Cells were released with DMSO, CDK2 inhibitor (CDK2i; 1 μM PF-07104091), CDK4/6i or CDK2i + CDK4/6i (mean ± s.e.). n = 300 cells per condition; 1 out of 4 biological replicates. g, Top, schematic of E2F activation in cells released with DMSO versus CDK4/6i. Bottom, single-cell traces of mVenus intensity after release with growth medium + DMSO or CDK4/6i.
