Figure 5.

Doxycycline-inducible p53 expression system confirmed the established threshold of cell fate decision. (a and b) Flow cytometric analysis of apoptosis by Annexin V/7-AAD staining using tet-on-strict-p53-transfected MCF7 cells induced with 0–10 ng/ml doxycycline for 4 days. Tet-on-strict-GFP-transfected MCF7 cells were used as a control. Quantitation of the percentages of Annexin V-positive cells is shown in panel (b). Data are presented as mean±S.D. from three independent experiments. Unpaired Student’s t-test was conducted to determine the significance; *P<0.05, **P<0.01. (c and d) Western blotting analysis of p53 and apoptosis-associated proteins in MCF7 cells transfected with tet-on-strict-p53 plasmid and induced with 0.2–5 ng/ml doxycycline for the indicated duration. Quantification of p53, BAX, and PUMA protein abundance is shown in panel (d). Shaded areas indicate the cumulative level of p53 at corresponding time points. (e and f) Model illustrating how E∫p53 controls cell fate. Prolonged low-dose treatment of Dox initiated a series of pulses followed by a terminal pulse with increased amplitude (e). Acute and high-dose Dox treatment induced a sustained accumulation of p53 (f). E∫p53, the cumulative level of p53 above a MEL, discriminates the activation of different sets of target genes and thereby differentiates cell fate choice. NS, not significant