Cells were plated in a 24-well glass-bottom imaging plate (Cellvis, USA) and cultured in a phenol red–free CO2-independent medium (Invitrogen) supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cell images were acquired with a Nikon TE2000-PFS inverted microscope enclosed in a humidified chamber maintained at 37°C. Cells were imaged every 10 min using a motorized stage and a 20× objective (numerical aperture, 0.95).

For data plotted in Figs. 1 and 5, we viewed and analyzed the images manually to determine the drug response phenotypes using the MetaMorph software (Molecular Dynamics). We scored cell fate by morphological tracking as follows: interphase (by flat morphology), entry into mitosis (by cell rounding), cell division (by respreading and splitting), cell cycle arrest (by absence of cell division for 72 hours), and cell death (by blebbing followed by cell lysis). The dynamic mode of nuclear p53 was scored on the basis of the p53-Venus fluorescence in the nucleus. To quantify the single-cell p53 trajectories, we used an automatic cell tracking program that we developed using MATLAB. The program consists of image analysis procedures that sequentially segment the individual cells, track them in time, identify the nucleus, and measure the p53 fluorescence intensity in the nucleus.

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