Fig. 4. The canonical signaling order of Rb inactivation before APC/C^CDH1 inactivation is reversed in cells proliferating without CDK4/6 activity.

a Cells mitogen released with DMSO or 1 µM CDK4/6 inhibitor and have recently inactivated APC/C^CDH1 were analyzed for phospho-Rb(S807/S811) signal (>800 cells per histogram for DMSO > 200 cells per histogram for CDK4/6 inhibitor; 1 of n = 3 biological replicates). b Top: Single-cell mRNA quantification (scale bar = 10 µm). Bottom: E2F1 mRNA in cells released with or without CDK4/6 inhibitor. High cyclin E/A-CDK activity defined as ratio of signal >0.7. p values calculated using two-sided, two-sample t tests (from left to right: n = 784, 1484, 638, and 98 cells). Box center indicates median, box edges denote quartiles, and whiskers extend to 1.5 times the interquartile range away from the box edges. One of n = 3 biological replicates. c Mitogen-released cells were assayed for chromatin-bound Rb. Green arrows: cells that have inactivated both Rb and APC/C^CDH1; blue arrows: cells that have inactivated Rb but not APC/C^CDH1; orange arrows: cells that have inactivated APC/C^CDH1 but not Rb. Scale bar: 10 µm. One of n = 2 biological replicates.