Extended Data Fig. 9: Functions of T373 site in Rb are tested in the Rb mutant re-expression assay.
From: An intermediate Rb–E2F activity state safeguards proliferation commitment
Related to Fig. 5. a, b, Histograms of total-Rb intensity (a) and E2F activity traces (b) in cells with or without RB1 knockdown or with RB1 knockdown + exogenous Rb expression. Cells were fixed 36 h after release with growth media + CDK4/6i. Cells were treated with si-RB1 one day before release to knockdown endogenous Rb and treated with Dox 5 h before release to induce exogenous Rb. A threshold for Rbhigh is Rb > 211 (n = 435, 537, 368, 173 cells for si-Control, si-RB1, si-RB1 + Rb-O.E., si-RB1 + Rbhigh, respectively. E2F: mean ± SE). c, single-cell traces of E2F activity in cells expressing the Dox-inducible HA-tagged Rb constructs. Cells were released with growth media + CDK4/6i. Cells were treated with si-RB1 one day before release to knockdown endogenous Rb and treated with Dox 5 h before release to induce exogenous Rb. To compare the same expression levels of Rb constructs, cells with 210 < HA < 211 were gated for analysis (black lines denote the example single-cell traces of 20 cells from one representative experiment. red lines denote the mean of a whole population. n = 323, 288, 383, 246 cells for Rb-WT, Rb-WT-3A, Rb-∆CDK, Rb-∆CDK-3D, respectively. n = 4 biological replicates for Rb-WT, Rb-WT-3A, Rb-∆CDK, and 3 biological replicates for Rb-∆CDK-3D).
