a, An example of cell phase gating using Hoechst and 5-ethynyl-20-deoxyuridine (EdU) staining. b, c, Box plot of nuclear 53BP1 (b) and γH2AX (c) puncta area in different cell-cycle phases with and without addition of a 20-min NCS (200 ng ml−1) pulse 8 h before fixation. Boxes reflect the 25th and 75th percentiles, whiskers denote the total range (53BP1; n > 2,800, γH2AX; n > 3,000 cells for each condition). d, Representative images of γH2AX and 53BP1 staining 8 h after a pulse of NCS (200 ng ml−1) for 20 min. Scale bar, 20 μm. e, Classification of CDK2inc and CDK2low cells after mitosis. f, Plot of nuclear γH2AX puncta area as a function of time relative to mitosis in unperturbed cells. Data are mean ± s.e.m. (n = 4 biological replicates). g, Histogram of nuclear Rb (Ser807/S811) intensity. h, Comparison of the E2F1 mRNA abundance by nuclear Rb (Ser807/S811) status (E2F1 is an E2F target). Data are mean ± s.d. (n = 10 wells (each well; n > 500 cells)). i, After selecting cells based on time since mitosis, G0/G1 cells were gated using Hoechst and EdU staining (top), and then cells in G0/G1 were further classified into hypo- or hyper-Rb population (bottom). j, Bar graph of nuclear γH2AX and 53BP1 puncta area. Data are mean ± s.e.m. (control; n = 4, NCS; n = 3 biological replicates). P values were calculated by unpaired two-tailed t-tests.