Fig. 1: Quantitative cell-cycle-resolved single-cell tracking based on endogenous markers.

a, Representative images from time-lapse microscopy with multigenerational cell tracking of mEGFP–53BP1 U-2 OS cells. The daughter cell generation (F₁) and granddaughter cell generation (F₂) generations with 53BP1 foci after cell division are highlighted. Scale bars, 50 µm. b, Example cell lineage depicting lineage relationships and DNA damage marked by 53BP1. P, parental cell generation; M₁, first mitosis; M₂, second mitosis. Time-lapse microscopy for 65 h at 30 min intervals. c, High-content-microscopy-derived EdU profiles of the parental and the edited 53BP1–PCNA U-2 OS cell line. a.u., arbitrary units. d, Representative images of endogenously tagged 53BP1 from 53BP1–PCNA U-2 OS cells treated as indicated and fixed after 45 min. Scale bars, 10 µm. e, Representative images from 53BP1–PCNA U-2 OS cells depicting endogenous PCNA patterns during S phase. Scale bars, 10 µm. f, Representative images of the cell-tracking procedure, depicting the smoothened endogenous PCNA signal for nuclei segmentation, the nuclear mask and the single-cell tracks applied on the non-smoothened PCNA signal. Scale bars, 10 µm. g, Single-cell lineages of the cells depicted in f. Endogenous PCNA patterns are depicted according to the colour code indicated. h, The same single-cell lineages as in g, with endogenous PCNA foci intensities depicted according to the colour code indicated. i, Single-cell lineages from endogenously tagged 53BP1–PCNA U-2 OS cells. PCNA foci are depicted according to the colour code indicated. j, The same single-cell lineages as in i, with cell cycle phase of the F₁ generation, based on the endogenous PCNA signals, depicted according to the colour code indicated (G1, S, G2). k, The cell cycle distribution derived from the time spent in G1, S and G2 according to the endogenous PCNA foci pattern of the single-cell lineages shown in i and j. Representative single-cell images are shown below. Scale bar, 10 µm.