Fig. 1. Acute CDK4/6 inhibition reveals a delayed and less efficient cell-cycle entry mechanism.

a Two mechanisms of starting the cell cycle. b Left: Degron-based APC/C activity reporter. Right: Mitogen-starved MCF-10A cells expressing the APC/C activity reporter were imaged at the time of mitogen and 1 µM Palbociclib (CDK4/6 inhibitor) addition. Time points taken every 12 min. CDK4/6 inhibitor refreshed at 24 h. Mean ± SEM from five biological replicates. Vertical gray dashed line: time of refreshing drug. c MCF-10A cells mitogen released in the presence of EdU with or without CDK4/6 inhibitor. Cells were fixed after 24 h (26838, 36549, 29371 cells for unreleased, DMSO, and CDK4/6 inhibitor; 1 of n = 2 biological replicates). d Cells were mitogen released with or without indicated CDK4/6 inhibitor concentrations and the drug was refreshed every 12 h, 24 h, or not at all. Cells fixed at 36 h after mitogen release. S/G2 cells determined via EdU incorporation and DNA content. Mean ± SEM from three biological replicates; p values calculated using two-sided, two-sample t tests. e CDF of mitogen-released cells inactivating APC/C^CDH1. Only cells present at the time of mitogen release are tracked. n = 7521 and 9563 cells for DMSO and CDK4/6i, respectively. f Percent cells with inactivated APC/C in cells born into DMSO or CDK4/6 inhibitor. Cells aligned at birth. Mean ± SEM from five biological replicates. g Different cell lines treated with 1 µM CDK4/6 inhibitor for 48 h and refreshed at 24 h. Percent S/G2 defined as >2n DNA (determined by Hoechst) and high endogenous geminin (an APC/C^CDH1 substrate). Percentages normalized to the DMSO-treated condition. h Generation comparison of cell-cycle re-entry percentages in MCF-10A (Rb intact) and HeLa (Rb inactivated) cells (MCF10A: mean ± SEM from three biological replicates; p values calculated using two-sided, two-sample t tests).