Differential p53 dynamics as a function of DNA-damage strength. (a) Phospho-H2A.X levels (DNA damage marker) in U-2 OS cells treated with increasing doses of etoposide for 12 hours. Actin served as a loading control. (b) Induction kinetics of p53-Venus and endogenous p53 in the clonal U-2 OS report line treated with 1 and 100 μmol/l etoposide at the indicated time points (in hour). The upper band is p53-Venus (around 80 kDa) and the lower band is endogenous p53 (approximately 53 kDa). (c) Single-cell trajectories of p53 dynamics at the nucleus quantified from p53-Venus fluorescence. U-2 OS cells were treated with either 1 μmol/l (black lines) or 100 μmol/l (red lines) of etoposide at time 0. Individual cells were tracked for 72 hours or until cell death occurred. The abrupt end of p53 trajectories before 72 hours corresponds to the time of death.