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Figure S1. Identification of differentially-regulated p53 target genes between P72 and R72 NHFs
using microarray experiment
(A) Schematic demonstration of the experimental design. Two separate sets of NHFs with homozygous
P72 or R72 variant of p53 were treated with 5Gy γ-irradiation for 2 or 4 hours. Total RNA was extracted
with Trizol and applied on the human gene expression array. One pair of NHFs with short-hairpin RNA
against p53 (shP53) was included to ensure that the identified expression changes were p53-dependent.
(B) Representative heatmap for the microarray experiment. Genes shown were altered more than 50% by
γ-irradiation compared to untreated samples with p-value smaller than 0.01.
(C) Ingenuity pathway analysis of genes identified in the microarray experiment. Upper panel: Cellular
functions and diseases associated with genes commonly regulated by P72 and R72 p53; Lower panel:
Cellular functions and diseases associated with genes differentially regulated between P72 and R72 p53.
(D) p53-upregulated genes identified by the microarray. Red circle: Genes that are upregulated more than
50% in R72 cells; Purple circle: Genes that are upregulated more than 50% in P72 cells. Fifty-seven
genes are upregulated by >50% in both P72 and R72 cells (black-dashed area). Thirty-three, and one,
gene(s) are upregulated specifically in P72 and R72 cells, respectively (>50% induction in one variant but
<5% alteration in the other).
Figure S2. p53-dependent TRIML2 induction is increased in R72 cells and positively regulates p53
activation and apoptosis
(A) p53-dependent, R72-specific TRIML2 induction as shown on the microarray heatmap.
(B) The osteosarcoma cell line Saos2 containing temperature-sensitive versions of P72 and R72 (with
V138A mutation) were incubated in 39˚C (inactive p53) or 32˚C (active p53) for 24 hr. Total RNA were
extracted and analyzed by qRT-PCR for the induction of TRIML2 mRNA, relative to control (cyclophilin
A). The gene expression level in 39˚C-incubated P72 cells was set to 1-fold and the data are normalized
to cyclophilin A. The data depicted are the averaged results from three independent experiments; error
bars mark standard error. The double asterisk (**) denotes p<0.005.
(C) Left panel: Wild-type (WT) or p53-knockout (p53-/-) human colorectal cell line Hct116 were treated
with 5μM 5-FU for 24 hr and whole cell lysates were subjected to western blot analysis to detect p53,
TRIML2 and GAPDH (loading control); Right panel: WT and p53-/- Hct116 cells were treated with
10J/M2 UV for 0, 1, 4, and 8 hr. Whole cell lysates were subjected to western blot analysis to detect p53,
TRIML2 and GAPDH (loading control).
(D) Western blot analysis of TRIML2 and cleaved caspase 3 (CC3, marker of apoptosis) in Hct116 cells
infected with control vector or sh-B to TRIML2, treated with 5-FU (5μM) for 72 hr. The data depicted are
representative of three independent experiments.
(E) U2OS cells stably transfected with control vector (pLKO1) or TRIML2 shRNA were left untreated or
treated with 5μM 5-FU for 24 hr. Whole cell lysates were subjected to western blot analysis to detect
p53, TRIML2, p21, Cleaved lamin A (CLA, apoptosis marker), and GAPDH (loading control). Signal
intensities were quantified using ImageJ software and normalized to GAPDH. Signals in U2OS cells with
TRIML2 shRNA were set to 1.
(F) WT, p53-/-, and TRIML2-overexpressing (WT-TRIML2) Hct116 cells were treated with DMSO or
100μM etoposide for 24 hr. Whole cell lysates were subjected to western blot analysis to detect p53,
TRIML2, p21, Cleaved caspase 3 (apoptosis marker), and GAPDH (loading control). Signal intensities
were quantified using ImageJ software and normalized to GAPDH. The cleaved caspase 3 signal in
etoposide-treated WT cells was set to 1.
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Figure S3. TRIML2 contributes to the different apoptotic potential between P72 and R72
(A) H1299 cells with Tet-inducible p53 of P72 or R72 variant were stably transfected with control vector
or TRIML2-overexpression construct and selected for with G418. Doxycycline and etoposide were used
to induce p53 expression and activation, respectively. Whole cell lysates were subjected to western blot
analysis to detect p53, TRIML2, PARP (poly ADP ribose polymerase; cleaved form indicated by the
arrow is an apoptosis marker), Cleaved caspase 3 (apoptosis marker), and GAPDH (loading control).
Signal intensities were quantified using ImageJ software and normalized to GAPDH. The levels of
cleaved PARP and cleaved caspase 3 in vector-control, doxycycline/etoposide-treated cells was set to 1fold.
(B) H1299 cells with Tet-inducible p53 of P72 or R72 variant were stably transfected with control vector
or TRIML2 shRNA and selected for with puromycin. Doxycycline and etoposide were used to induce
p53 activation and whole cell lysates were subjected to western blot analysis to detect p53, PARP (poly
ADP ribose polymerase; cleaved form indicated by the arrow is apoptosis marker), Cleaved lamin A
(apoptosis marker), and GAPDH (loading control). Signal intensities were quantified using ImageJ
software and normalized to GAPDH. The levels of cleaved PARP and cleaved lamin A in vector-control,
doxycycline/etoposide-treated cells was set to 1-fold.
Figure S4. Effect of TRIML2 knockdown on p53 target gene expression
(A, B) Hct116 cells stably transfected with control vector (pLKO1) or TRIML2 shRNA were treated with
DMSO or 5μM 5-FU for 72 hr. Total RNA were extracted and subjected to qRT-PCR to detect the
mRNA levels of TRIML2 and p53 target genes that are known to be associated with (A) TA2
(transactivation domain-2)-mediated tumor suppression, and (B) metabolism. The expression of each
gene in vector-control, DMSO-treated cells was set to 1-fold. The data depicted are the averaged results
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from three independent experiments, normalized to control (cyclophilin A); error bars mark standard
error. The asterisk denotes a p-value <0.05.
(C) Summary of TRIML2-regulated p53 target genes. Genes are considered significantly altered by
TRIML2 shRNA if their expression levels are 1) upregulated by 5-FU in control cells by >50%, and 2)
altered by more than 20% following TRIML2 knockdown compared to control/5-FU (p-valve < 0.05).
(D) p53 target genes that are positively regulated by TRIML2 (reduced induction following TRIML2
knockdown).
(E) p53 target genes that are negatively regulated by TRIML2 (increased induction following TRIML2
knockdown).
Figure S5. TRIML2 regulates a subset of p53 target genes
(A) Hct116 cells stably transfected with control vector (pCDNA3) or TRIML2-expression construct were
treated with DMSO or 5μM 5-FU for 72 hr. Total RNA was extracted and subjected to qRT-PCR to
detect the mRNA levels of TRIML2 and p53 target genes. The expression of each gene in TRIML2overexpressing, DMSO-treated cells was set to 1-fold. The data depicted are the averaged results from
three independent experiments, normalized to cyclophilin A; error bars mark standard error. The asterisk
denotes a p-value <0.05. (B) qRT-PCR analysis of the levels of Bax, Bbc3 (Puma), and PIDD in Saos2
cells containing temperature-sensitive versions of P72 and R72 (with V138A mutation), following
incubation in 39˚C (inactive p53) or 32˚C (active p53) for 24 hr. The level of each gene in 39˚Cincubated P72 cells was set to 1-fold and the data are normalized to cyclophilin A. The data depicted are
the averaged results from three independent experiments; error bars mark standard error. The double
asterisk (**) denotes p<0.005.
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Figure S6. TRIML2 does not affect SUMO1- or Ubiquitin modification of p53; TRIML2 is the only
TRIM protein differentially regulated by codon 72 polymorphism of p53.
(A) Immunoflourescence analysis in H1299 cells cotransfected with p53 and TRIML2. Twenty-four
hours after transfection, cells were fixed with paraformaldehyde and p53 (green) and TRIML2 (red) were
detected and imaged by confocal microscopy. DAPI counterstain (blue) was used to stain the nucleus.
Yellow staining in the overlay image indicates colocalization of p53 and TRIML2 (enlarged image on the
right).
(B) Western blot analysis for p53 and indicated proteins in H1299 cells transfected with TRIML2, SUMO1, and p53, along with the positive control genes indicated (TRIM27, PML). The right brace denotes the
higher-molecular weight species of p53 that are induced.
(C) Western blot analysis for p53 and indicated proteins in H1299 cells transfected with TRIML2,
Ubiquitin (Ub), and p53, along with the positive control genes indicated (TRIM27, PML). The right brace
denotes the higher-molecular weight species of p53 that are induced.
(D) qRT-PCR analysis of the levels of different TRIMs (TRIM8, TRIM13, TRIM19, TRIM22, TRIM24,
TRIM29) in H1299 cells with tetracycline-inducible P72 or R72 (treatment with 0.75 μg/mL doxycycline
plus 100 μM etoposide for 24 hr) and Saos2 cells containing temperature-sensitive versions of P72 and
R72 (incubation in 39˚C or 32˚C for 24 hr). All the data are normalized to cyclophilin A. The data
depicted are the averaged results from three independent experiments; error bars mark standard error.
Table S1. Genes specifically upregulated by P72 or R72 p53 in microarray analysis
Table S2. Primers used for qRT-PCR and ChIP assays
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