Fig. 6. APC/C^CDH1 inactivation remains reversible until Rb inactivation in cells lacking CDK4/6 activity.

a Cells born into DMSO or 1 µM CDK4/6 inhibitor were treated with 10 µM EdU for 15 min, and then assayed for EdU and phospho-Rb(S807/S811) (5000 cells plotted, 1 of n = 3 biological replicates). b Mitogen-released cells treated with DMSO or CDK4/6 inhibitor were measured for percent cells with high phospho-Rb(S807/S811) signal, high APC/C degron, and high EdU signal (after 15 min of 10 µM EdU treatment). Mean ± SEM from three biological replicates. c Emi1 ensures irreversible APC/C^CDH1 inactivation. d–h Cells born into DMSO or CDK4/6 inhibitor and have inactivated APC/C^CDH1 (with the exception of (f), where it is just cells born into DMSO or CDK4/6 inhibitor) were treated with 3 µM CDK1/2i (d, e) or an extra 100 mM salt (f–h). Dashed lines denote 25th and 75th percentile and bottom row panels are plotted in log₂ scale for better visualization. APC/C degron detected via signal over background. Gray boxes denote conditions where cells reactivated APC/C^CDH1. n = 2 biological replicates; n > 30 cells per condition. In g, h, to enrich for Rb inactivated cells in the 5 h post APC/C^CDH1 degron detection conditions, only cells with cyclin E/A-CDK >0.8 right before treatment were analyzed (threshold determined from Fig. 3e).