Supplementary Information
Materials and Methods
Cell culture and treatment
Primary mouse embryonic fibroblasts (MEFs) were harvested from E13.5 days embryos
and thymocytes were harvested from 4-5 weeks old mice and cultured in 10% FCS
containing DME medium as described previously 1.
Cell viability was analyzed by staining with annexin-V and propidium iodide, as
described 2, after treatment with UV or -irradiation, Thapsigargin (1M), hydrozyurea
(2mM) or nutlin (10M). For determining BrdU+ cells, cells were labeled with BrdU
(10M) 1hr prior to harvesting and stained with FITC-conjugated anti-BrdU antibody
(BD Biosciences). Cells were analyzed by flow cytometry using the WinMDI software.
Immunoblot analysis
Whole cell lysates and tissue homogenates were prepared with NP-40 lysis buffer as
described 2. Nuclear extract were prepared with NE-PER Nuclear and Cytoplasmic
Extraction Reagents (Pierce) according to manufacturer’s instruction. 50-100g protein
of whole cell lysate/tissue homogenate or nuclear extract was separated by 10% SDSPAGE for subsequent Western blot analysis with anti-phospho-p53 (Cell Signaling), antip53 (1C12, Cell Signaling; DO-1, Santa Cruz), anti-cyclin D (Santa Cruz), anti-actin
(Sigma), anti-tubulin (Cell Signaling) and anti-lamin A/C (Cell Signaling) antibodies. To
determine the half-life of p53 protein, cells were treated with 50μg/ml of cycloheximide
and harvested at different time points. For detecting ubiquitinated p53, ubiquitinated
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proteins were enriched from whole cell lysate using a ubiquitin enrichment kit (Pierce) as
per manufacturer’s instructions, followed by immunoblot analysis.
Quantitative real-time RT-PCR of p53 target genes and microarray analysis
Total RNA was prepared and quantitative RT-PCR of p53 target genes were performed
using gene-specific primers, as described 2. Fold induction was calculated with reference
to the untreated samples. For microarray analysis, total RNA was extracted from the
lymph nodes of lymphoma bearing mice and Affymetrix GeneChip® mouse exon 1.0ST
arrays were used. The data was analyzed with Partek genomic suite software (Partek)
and ArrayAssist® Expression Software (trial version, Stratagene).
Fluorescence immunocytochemistry
Cells were fixed with 4% paraformaldehyde and stained with anti-tubulin (Sigma) or
anti-p53 (Pab240, Calbiochem) antibodies followed by Alexa488-conjugated anti-mouse
IgG antibody (Invitrogen).
Stained cells were mounted with Vectashield mouting
medium containing DAPI (Vector laboratories) and analyzed by fluorescence
microscopy. Cell numbers and centrosome numbers were manually counted in a blinded
manner (without knowing the genotypes). 2 independent experiments with 3 clones of
MEFs in each experiment were performed.
Statistical analysis
The following tests were utilized for statistical analysis: Chi-square test to detect
differences between expected and obtained offsprings; Unpaired t-test for analysis of
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BrdU-incorporation differences; Non-linear regression was was used to determine p53
half-life differences; 2-way ANOVA for analysis of differences in centrosome analysis;
and Log-rank (Mantel-Cox) test for median survival of various groups of mice in longterm tumor formation studies.
Supplementary Figure Legends
Supplementary Figure 1. Amino acid spanning the S315 residue of human p53 is
highly conserved
(A)
Multiple sequence alignment of p53 was performed with human, rhesus, cow,
sheep, pig, guinea pig, mouse, rat and Chinese hamster protein sequences. The epitope
neighboring S315 residue of human p53 protein is highlighted by the box.
(B)
p53 null MEFs were transfected with wild-type or S312A p53 expressing
plasmids and treated with 1M thapsigargin (TG) for the indicated time. Whole cell
lysates were subjected to immunoblot analysis. N.S.: non-specific bands are indicted
with arrows.
Supplementary Figure 2.
(A)
Analysis of p53 protein in MEFs, thymus and liver
p53+/+ and p53S312A/S312A MEFs and thymocytes were -irradiated (IR) at 10Gy
and 5Gy respectively, and the p53 status was analyzed by Western blotting at various
time points as indicated. p53-/- cells served as negative controls. N.S.: non-specific bands
are indicted with arrows.
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(B)
Wild-type mice of 3 weeks old or 5 months of age were -irradiated (10Gy) and
livers were harvested at the indicated time points. p53-/- mouse liver was also harvested as
a control. p53 level were determined by Western blotting.
Supplementary Figure 3. S312A MEFs display normal cell cycle kinetics
(A)
Asynchronous, unstressed MEF cultures of various p53 genotypes were pulsed
with 10M BrdU for 1hr and harvested at the indicated times after the BrdU wash off
(1hr after pulse). The % of total BrdU+ cells are indicated in the each box.
(B)
The % of BrdU+ cells having 2N or 4N DNA content was determined from the
data shown in (A) by flow cytometry.
(C)
mRNA from p53+/+ and p53S312A/S312A MEFs (4-5 independent clones/genotype)
were used for analysis of p53 target genes (noxa, p21 and mdm2) and cyclins by qRTPCR and normalized against gapdh expression. Data represent mean±SD.
Supplementary Figure 4.
Microarray analysis of E-myc transgene induced B-cell
lypmyhoma
The volcano plot of p-vaule against fold change (FC) obtained from microarray analysis
by comparing the lymphoma samples of E-mycTg; p53S312A/S312A and E-mycTg; p53+/+
mice. Each square represent 1 expressing gene in the microarray analysis.
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References
1.
Lee MK, Sabapathy K. Phosphorylation at carboxyl-terminal S373 and S375
residues and 14-3-3 binding are not required for mouse p53 function. Neoplasia
2007; 9(9): 690-8.
2.
Lee MK, Sabapathy K. The R246S hot-spot p53 mutant exerts dominant-negative
effects in embryonic stem cells in vitro and in vivo. J Cell Sci 2008; 121(Pt 11):
1899-906.
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