Fig. 2: Alterations in Rb-protein levels depending on cell proliferation.

a Immunoblot showing exogenous and endogenous Rb and GAPDH expression in MCF-10A and RPE1 cells after treatment with palbociclib (1 µM). b Relative levels of endogenous and exogenous Rb protein. Solid lines indicate best-fitted lines. Rb half-life is shown as the mean ± SD (n = 2 biological replicates). c Single-cell traces of CDK2 activity and YFP-Rb levels and mean YFP-Rb levels aligned by the end of mitosis (anaphase) in MCF-10A cells. Based on CDK2 activity (threshold = 0.5, black line) between 9 and 15 h after mitosis, daughter cells were classified into proliferation or quiescence. Mean YFP-Rb levels are shown as mean ± 95% confidence interval (proliferation: n = 64 cells; quiescence: n = 72 cells). d Time course of relative YFP-Rb level change in MCF-10A cells entering quiescence. YFP-Rb levels were normalized with the minimum level (n = 72 cells). e Heatmap of single-cell traces for CDK2 activity and YFP-Rb levels in MCF-10A cells treated with palbociclib (1 µM) + doxycycline (5 µM). Each row represents a single-cell trace over time according to the respective color map. During 25‒40 h after drug treatment, cells activating CDK2 (>0.8) for over 2 h were classified into proliferating cells. f Time course of relative YFP-Rb level change in MCF-10A cells treated with cycloheximide (10 µg/ml). Cells were treated with either DMSO or palbociclib (1 µM) for 24 h before imaging (DMSO: n = 3613 cells; CDK4/6i: n = 2717 cells). g Time course of relative YFP tagged wild-type and phosphomimetic Rb in MCF-10A cells treated with cycloheximide (10 µg/ml). Cells were treated with palbociclib (1 µM) for 24 h before imaging (WT: n = 281 cells; phosphomimetic: n = 128 cells).