Extended Data Fig. 7: Preferential phosphorylation of Rb at T373 is generalizable in cycling cells, and slower dephosphorylation at T373 in Rb compared to other sites creates partially phosphorylated Rb/intermediate E2F activity states in cycling cells.

Related to Figs. 3 and 5. a, Left, Rb phosphorylation at T373 after anaphase measured by immunofluorescence. Cells were asynchronously cycling in starvation media + EGF (0.2 ng/mL). Pseudo-time course immunofluorescence was measured by computationally aligning cells at anaphase based on live-cell images. Cells were color-coded based on Rb phosphorylation at T373 (green: Rb phosphorylation at T373  0.6, 0-5 h after anaphase, blue: Rb phosphorylation at T373  0.6, 5-15 h). A cyan line shows median levels of Rb phosphorylation at T373 = 1.0 (n = 3 biological replicates. n = 1719 cells in total). Right, representative single-cell traces of E2F activity categorized in Left (n = 3 cells each. 1 of n = 3 biological replicates). b, Model for three different cell fates based on Rb phosphorylation across cell generations. The background color code is matched with the categorization in a. (i) Daughter cells are born with partially phosphorylated Rb involving p-T373 (green) and dephosphorylate Rb completely to undergo quiescence (blue). (ii) Daughter cells are born with partially phosphorylated Rb involving p-T373 (green) and hyperphosphorylate Rb to enter S phase (orange). (iii) Daughter cells are born with hyperphosphorylated Rb and enter S phase quickly (magenta). c, Rb dephosphorylation kinetics after anaphase at T373 (left) and S807/S811 (right) measured by immunofluorescence. Cells were asynchronously cycling in starvation media + EGF (0.2 ng/mL). Cells were treated with or without 50 ng/mL neocarzinostatin (NCS) for 20 min, and incubated in starvation media + EGF (0.2 ng/mL) again for 16 h before fixation. Pseudo-time course immunofluorescence was measured by computationally aligning cells at anaphase based on live-cell images. Cells that were born 1 h to 16 h after NCS or mock treatment were analyzed. A cyan line shows median levels of Rb phosphorylation <0.6 and a red dotted line shows a reference line of Rb phosphorylation = 1.0 (n = 3 biological replicates. n = 1719, 1160 cells in total for NCS (−), NCS (+), respectively). d, Phosphorylation Site Preference (PSP) plots showing single-cell correlation of Rb phosphorylation at T373 plotted against S807/S811. Each phosphorylation signal was normalized by the total Rb antibody signal in the same cell and each axis was adjusted to the average phosphorylation signal in S phase of 1. Color indicates relative cell population density. A red line shows fitting with a preferential relative phosphorylation or dephosphorylation rate between the two sites (PSP_coeff) (see more details in Methods). Cells asynchronously cycling in starvation media + EGF (0.2 or 20 ng/mL) were fixed 16 h after 50 ng/mL neocarzinostatin (NCS) treatment for 20 min or mock treatment (n = 3393, 1904, 3079, 2247 cells for NCS (−) in EGF (0.2 ng/mL), NCS (+) in EGF (0.2 ng/mL), NCS (−) in EGF (20 ng/mL), NCS (+) in EGF (20 ng/mL), respectively. 1 of n = 3 biological replicates). e, Box plots of 53BP1 puncta area. Box centers are median values, box edges are the 25th and 75th percentiles, and whiskers are minimum and maximum values. Cells asynchronously cycling in starvation media + EGF (0.2 or 20 ng/mL) were fixed 16 h after 50 ng/mL neocarzinostatin (NCS) treatment for 20 min or mock treatment. p-values were calculated using two-sided, two-sample t tests (n = 100 cells each, p-value (NCS (−) vs (+) in EGF (0.2 ng/mL)) = 2.0 × 10⁻¹⁴. p-value (NCS (−) vs (+) in EGF (20 ng/mL)) = 2.5 × 10⁻¹⁸. 1 of n = 2 biological replicates).