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. Author manuscript; available in PMC: 2020 Apr 29.
Published in final edited form as: Mol Cell. 2019 Sep 19;76(4):562–573.e4. doi: 10.1016/j.molcel.2019.08.020

Figure 1. Heterogeneity in Cyclin E/A-CDK Activity Obscures Regulatory Mechanisms of Rb Hyperphosphorylation in Bulk-Cell Analysis.

Figure 1.

(A) Schematic highlighting possible threshold or cell-cycle timing-dependent maintenance of Rb hyperphosphorylation by cyclin E/A-CDK following acute CDK4/6 inhibition.

(B) MCF-10A cells mitogen-released for indicated times prior to harvesting and western blot analysis. For 12 and 18 h time points, cells were treated with palbociclib 1 μM or vehicle (0.1% DMSO) for 15 min prior to harvesting.

(C) Schematic of cyclin E/A-CDK activity reporter.

(D) MCF-10A expressing cyclin E/A-CDK activity reporter and H2B-mTurquoise (for tracking) were mitogen-released and live-cell-imaged. Left: 50 random traces with automated detection of initial activation shown for illustration. Right: percentage of cells that have started to activate cyclin E/A-CDK by a given time since mitogen release. Error bars are SD; n = 3 replicates; n ≥ 105 cells per replicate.