SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1. Doxycycline-inducible p53 is phosphorylated on Ser15 and Ser46
and acetylated on Lys382.
(a) Immunoblot analysis of p53, phospho-p53 (Ser15 and Ser46) and acetyl-p53 (Lys382) in
B5/589-p53 cells induced with 1-10ng/ml doxycycline for 24h. Parental B5/589 cells were
treated with 0.5µM or 1 µM doxorubicin for 24h. (b) Cells treated as in (a) were harvested in 1%
SDS lysis buffer to isolate the nuclear fraction, and probed for γH2AX, phospho-p53 (Ser15) and
p53. (c) Immunoblot analysis of p53 and phospho-p53 (Ser15 and Ser46) in B5/589 cells that
were treated with 5ng/ml doxycycline and in B5/589-p53 cells induced with 5ng/ml doxycycline
for 24h. 20µg and 100µg of protein lysates were loaded in order to detect p53 protein and
phosphorylation levels of endogenous p53 in B5/589 cells. (d) Immunoprecipitation of p53 with
p53 or control IgG antibody, followed by immunoblotting with phospho-p53 (Ser15 and Ser46)
and p53 antibody. B5/589-p53 cells were induced with 3ng/ml or 5ng/ml doxycycline for 24h.
200µg or 800µg of protein lysates were immunoprecipitated in an effort to match 3ng/ml and
5ng/ml doxycycline-induced p53 levels (see lane 7 and 8). (e) Immunoblot analysis of p53,
phospho-p53 (Ser15 and Ser46) and acetyl-p53 (Lys382) in B5/589-p53 cells induced with
5ng/ml or 10ng/ml for 24h. Parental B5/589 cells were transduced for 72h with four lentiviral
vectors containing p53 mutants that cannot be phosphorylated on Ser15 or Ser46, or acetylated
on Lys120 or Lys382 (designated S15A, S46A, K120R and K382R). β-actin was used as a
loading control in (a-c) and (e).
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Supplementary Figure S2. Overexpression of p53 at super-physiological levels accelerates
apoptosis.
(a) Flow cytometry analysis of DNA content by PI staining of B5/589-p53 cells induced with the
indicated levels of doxycycline (DOX) for 2 days. Representative flow cytometry profiles and
percentages of cells in sub-G1 (<G1), G1 and S phases are shown. (b) Flow cytometry analysis
of apoptosis by Annexin V/PI staining of the same B5/589-p53 cell described in (a).
Representative flow cytometry profiles and percentages of ‘Living’ cells (Annexin V and PI
negative cells in lower left quadrant) and ‘Annexin V’ positive cells (combined percentages of
Annexin V positive cells in lower right and upper right quadrants) are shown. (c) Flow cytometry
analysis of DNA content by PI staining of parental B5/589 cells treated with the indicated levels
of doxorubicin. Representative flow cytometry profiles and percentages of cells in sub-G1
(<G1), G1, S and G2/M (G2) phases are shown. In agreement with previous studies, doxorubicin
treatment triggered G2/M arrest at lower doses and G2/M and S-phase arrest and cell death at
higher doses.25 (d) Immunoblot analysis of p53 expression levels in B5/589-p53 cells treated as
in (a). β-actin was used as a loading control. (d) Number of apoptotic cells positively correlates
with p53 protein levels. Annexin V positive cells from (b) were plotted against quantified levels
of p53 protein from (d).
Supplementary Figure 3. Cluster analysis of 21 pro-arrest and 21 pro-apoptotic genes.
21 pro-arrest and 21 pro-apoptotic genes from Figures 4d and 4e were grouped into 7 clusters
(A-G), according to the pattern of their expression. p53 gene was added for comparison. Gene
names in all clusters are shown: pro-arrest in green and pro-apoptotic in violet.
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Supplementary Figure 4. p53 levels in RKO and LNCaP cells determine the cell fate
decision between growth arrest and apoptosis.
(a-b) Flow cytometry analysis of DNA content by PI staining (a) or of apoptosis by Annexin
V/PI staining (b) of RKO cells incubated with low (3µM) or high (20µM) doses of nutlin-3 for 1,
2, 3, 4 and 7 days. (c-d) Flow cytometry analysis of DNA content by PI staining (c) or of
apoptosis by Annexin V/PI staining (d) of LNCaP incubated with low (3µM) or high (20µM)
doses of nutlin-3 for 1, 2, 3, 4 and 7 days.
Supplementary Figure 5. Lowering the apoptotic threshold with ABT-263 synergizes with
nutlin-3-mediated apoptosis in parental B5/589 cells.
Apoptosis in parental B5/589 cells measured by flow cytometry of Annexin V/PI staining. The
percentages of Annexin V positive cells are shown. Cells were incubated with low (3µM) or high
(20µM) doses of nutlin-3 (NT) and treated with 200nM or 500nM ABT-263 for 4 days. Cells
were also treated with 0.15% DMSO as a control. Data were fitted with a Boltzmann function.
Supplementary Figure 6. Threshold model for p53 dependent cell fate decision between
arrest and apoptosis.
p53 transactivates the same set of pro-arrest and pro-apoptotic target genes proportionally to its
expression levels. The biological outcomes of growth, arrest and apoptosis are determined by
distinct cellular thresholds at which arrest and apoptosis are executed.
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