Supplemental Information
Materials and Methods
Screening of a medaka cDNA library
To identify as yet unknown regulators of p53 and/or Mdm2 abundance, we performed a cell
culture based overexpression screen using a cDNA library from medaka early stage embryos
(Souren et al 2009) and p53-negative H1299 cells. The cDNA library contained about 18,000
open reading frames covering about 80% of the medaka genome. For the first round of
screening, H1299 cells were transfected with library pools, each pool consisting of 24
individual cDNAs, together with p53, Mdm2 and ROR2. The Wnt receptor protein ROR2 was
co-transfected as a non p53-related specificity control. SDS-PAGE and Western Blotting
analysis was then used to detect alterations in the abundance of p53 and Mdm2. Library
genes whose overexpression specifically altered the abundance of co-expressed p53 and/or
Mdm2 in at least two independent experiments were considered candidates. To identify the
individual clones that were responsible for the regulation of p53 and/or Mdm2 within the pool,
the single clones of the pool-hits were amplified from the cDNA library glycerol stock and
transfected into H1299 cells together with p53, Mdm2 and ROR2. Again, alterations in the
abundance of p53 and Mdm2 were detected by SDS-PAGE and Western Blotting. Clones
that specifically altered the abundance of co-expressed p53 and/or Mdm2 in at least two
independent experiments were considered candidates for regulators of p53 and Mdm2. One
of these genes was TRIM25. The regulation of p53 and Mdm2 by TRIM25 from medaka was
further confirmed by transfecting increasing amounts of a plasmid expressing medakaTRIM25 together with p53 and Mdm2 into H1299 and Western blot analysis. Another gene
that was identified in this screen was FUBP1, a known regulator of p53 and Mdm2
abundance (Jacob et al 2014, Liu et al 2011), validating the screen.
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Plasmids
Myc-ROR2 was constructed by PCR amplification.
Antibodies
The antibody against -7 was purchased from Enzo Life Sciences. The antibody against
HRP-conjugated anti-actin was from Cell signaling.
Mice
P53-negative mice (strain B6.129S2-Trp53tm1Tyj/J; The Jackson Laboratory) were crossed
with TRIM25-negative mice (Orimo et al 1999). The heterozygote offsprings were
intercrossed to receive single and double knock-out mice. The genotyping of the mice was
performed according to the suggestions of the suppliers of the knock-out mice.
Western blotting of tumor samples
Protein extracts, prepared from frozen tumor samples, were separated by SDS-PAGE and
the proteins were transferred onto nitrocellulose membranes. The membranes were blocked
with 10% non-fat dry milk in TBST buffer (10 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween
20) for 1h at room temperature and incubated with a rabbit polyclonal antiserum generated
against TRIM25 (1:250) and with an HRP-conjugated anti-actin antibody (1:1,000). The blots
for detection of TRIM25 were washed three times with TBST (5 min each wash) prior to
incubation with a 1:2,000 dilution of a HRP-conjugated secondary anti-mouse antibody for 30
min at room temperature. After three washing steps (5 min each) the blots were developed
with ECL (Amersham, Freiburg, Germany).
TRIM25 mRNA synthesis
Medaka TRIM25 was transcribed from linearized pCS2+-mdTRIM25-1 and pCS2+-mdTRIM25-2 using the mMESSAGE mMachine kit® (Ambion). Six base pairs of the target
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sequence of the MOs were replaced to make the RNA resistant to MOs. Sequences of
primers and MOs are available on request.
RT-PCR
Cells were lysed and total RNA was prepared using the RNeasy kit (Qiagen) according to the
manufacturer’s protocol. After DNAse I treatment, cDNA was synthesized using random
primers and m-MLV reverse transcriptase. The quality of cDNA was tested by conventional
PCR using primers for -actin. Quantitative real-time reverse transcription PCR (qRT-PCR)
was carried out in triplicates with SYBR® Green Real-Time PCR Master Mix (Qiagen) on an
ABI StepOnePlus System (Applied Biosciences). The cDNA was denatured for 4 min at 90°C
followed by 40 cycles of 90°C for 30s, 60°C for 45s and 72°C for 45s and a final extension
step of 5 min at 72°C. The transcript levels were normalised by the readings for -Actin.
For RT-PCR on medaka embryos, embryos at the desired dpf (days post fertilisation) were
lysed in Trizol (Invitrogen) and RNA was prepared according to the manufacturer’s
recommendations. Samples were treated with DNAseI for 30 min to remove contaminating
DNA. After inactivation of the DNAse, RNA was transcribed into cDNA using random primers
and RevertAidTMHMinusM-MuLV reverse transcriptase. PCR was performed using GoTaq
polymerase and primers for TRIM25, p53 and -Actin of medaka. After an initial denaturation
step, the cDNA was amplified in 40 cycles of 95°C for 30s, 60°C for 45s and 72°C for 45s
and a final extension step of 5 min at 72°C.
MTT assay
2 x 104 HCT116 wild-type or HCT116 p53-/- cells were plated into a 96-well plate and
transfected with TRIM25 siRNA or with a control siRNA. 3 days after plating, MTT was added
to the cells to a final concentration of 0.5 mg/ml for 4h. Thereafter, the medium was aspirated
and cells and the formazan salt were solubilised in isopropanol and measured at 550 nm
using an ELISA reader.
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DNA binding assay
The binding of p53 to target sequences was analysed by using the TF-Detect™ Human P53
Activity Assay Kit (GeneCopoeia) according to the manufacturer’s instructions. Therefore, 5 x
105 HCT cells were plated into a 6-well plate and transfected with 100 nM TRIM25 siRNA or
with a control siRNA. 2 days after transfection, cells were fractionated into cytoplasm and
nucleus and the protein concentration was determined. The wells of the assay plate (coated
with the p53-consensus binding site) were rinsed with 200 μl Wash Buffer. Then 50 μl
binding buffer A, supplemented with 1 mM DTT, and 25 μg of nuclear proteins were added to
the wells and incubated for 1 hour at RT with gentle rocking. The wells were washed three
times with 200 μl 1X Wash Buffer. Excess buffer was removed by tipping the top of the plate
onto absorbent paper. After washing, 100 μl of an anti-p53 antibody, that has been diluted
1:1000 in 1X binding buffer B, were added to each well and incubated for 1 hour at RT with
gently shaking. After incubation, the wells were washed three times with 200 μl 1X Wash
Buffer. Then the HRP-conjugated antibody was diluted 1:1000 in 1X binding buffer B. 100 μl
of this solution were added to each well and incubated for 1 hour at RT with gently shaking.
The wells were washed as described above. Finally, 100 μl TMB Substrate Solution were
added and incubated for 15-30 min until the color conversion was clearly visible. The
reaction was stopped by adding 100 μl of the Stop Solution and the color conversion was
determined at λ450 nm at an ELISA reader."
Sucrose gradient centrifugation
Cells were washed twice with ice-cold PBS, scraped into PBS, collected by centrifugation
and suspended in (50 mM Tris pH 7.4, 20 mM NaCl, 10 mM MgCl2, 0.5% NP40, 5 mM ATP,
1 mM DTT, 1 mM PMSF, 40 U/ml DNAse (Universal Nuclease for cell lysis; Pierce), 10 mM
N-ethylmaleimid, 10 mM 1,10-Phenanthtroline and 1x Phosphostop (Roche)). The lysate was
pushed 3 times through a 26G needle and incubated on ice for 30 min. The protein extract
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was cleared by centrifugation at 16,000 g at 4 °C for 15 min, loaded onto a 10–40% sucrose
gradient and centrifuged at 100,000 g at 4 °C for 18 h. Fractions were collected and analyzed
by Western Blotting.
Cell fractionation
Cells were washed with ice-cold PBS, scraped in PBS and collected by centrifugation. The
cellular pellet was suspended in homogenization buffer (10 mM HEPES pH 7.9, 10 mM KCl,
1.5 mM MgCl2, 0.5 mM DTT, protease inhibitors) and incubated for 5 minutes on ice. The
nuclei were released by 30 strokes with a douncer and collected by centrifugation for 5 min
at 300 g. The supernatant, which contained the cytoplasmic lysate, was transferred to a new
tube. The nuclei were suspended in 0.25 M sucrose buffer (0.25 M Sucrose, 10 mM MgCL2,
EDTA-Free Protease Inhibitor Cocktail (Tablets; Roche)) and layered onto a cushion of 0.88
M sucrose buffer (0.88 M Sucrose, 0.5 mM MgCL2, EDTA-Free Protease Inhibitor Cocktail)
followed by centrifugation at 2800 g for 10 min to clean the nuclei. The supernatant was
aspirated and the nuclei were lysed in NP40 lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5
mM EDTA, 1% NP40, 1 mM PMSF).
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Figure Legends for supplementary figures
Figure S1: TRIM25 increases p53 abundance. (A) H1299 cells were co-transfected with
increasing amounts of a plasmid encoding TRIM25, with plasmids encoding p53 and Mdm2
and with vector DNA to adjust for different amounts of DNA. 24h after transfection, cells were
harvested and abundance of p53, Mdm2 and TRIM25 was monitored by Western blotting.
Hybridisation with an antibody targeted against PCNA (proliferating cell nuclear antigen) was
performed for loading control. (B) MCF7 cells cultured in RPMI-medium (w/o phenol red)
containing 10% charcoal-stripped FBS were transfected with two different siRNAs targeting
TRIM25 or with a control siRNA. Cells were harvested at 72 hours after transfection. At the
indicated times prior to harvest, 10 nM β-estradiol was added. Abundance of p53, Mdm2,
TRIM25 and PCNA (for loading control) was monitored by Western blotting. The Western blot
signals were quantified and the ratios of p53 and PCNA, of Mdm2 and PCNA and of TRIM25
and PCNA were calculated.
Figure S2: TRIM25 does not alter the abundance of p53 and Mdm2 RNA. (A) H1299 cells
were co-transfected with plasmids encoding p53, Mdm2 and TRIM25 as indicated and with
vector DNA to adjust for different amounts of DNA. 24h after transfection, cells were
harvested. An aliquot of the cells was lysed and expression of p53, Mdm2 and TRIM25 was
determined by Western blotting. From the remaining cells, RNA was prepared and the
relative abundance of p53 and Mdm2 RNA was determined by qRT-PCR. Mean values and
standard deviations were calculated from the obtained 2^dCT values of 3 independent
experiments and blotted. Values of p53 in the absence of TRIM25 and Mdm2, and of Mdm2
in the absence of TRIM25 were set to 1. (B) MCF7 cells cultured in RPMI-medium (w/o
phenol red) containing 10% charcoal-stripped FBS were treated with 10 nM β-estradiol or
with vehicle. 24h after estrogen addition, cells were lysed and abundance of p53, Mdm2,
TRIM25 and PCNA (for loading control) was monitored by Western blotting. The Western blot
signals for p53, Mdm2 and PCNA were quantified and relative amounts of p53 and Mdm2
were calculated. Mean values and error rates of two independent experiments were
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calculated and plotted. Relative abundance of p53 and Mdm2 protein at the time of CHX
addition was set to 100%.
Figure S3: Association of TRIM25 with p53 and Mdm2. (A) MCF7 cells were lysed and
loaded onto a sucrose gradient. After centrifugation, 28 fractions were collected and
analysed for the abundance of p53, Mdm2 and TRIM25. Hybridisation with an antibody
directed against the proteasomal protein -7 was performed for internal control. Fractions
which contain all three proteins have been encircled. (B.I) MCF7 cells were fractionated into
cytoplasmic and nuclear lysate. Abundance of p53, Mdm2 and TRIM25 in these fractions
was determined by Western blotting. Hybridisations with antibodies against p300 (nuclear
protein) and -Actin (cytoplasmic protein) were performed for control. (B.II) MCF7 cells were
fractionated into cytoplasmic and nuclear lysate. 5% of the cellular lysate were used to
monitor expression of p53, Mdm2 and TRIM25 (Input). From the remaining cellular lysate,
TRIM25 was immunoprecipitated. Precipitation with IgG was performed for control.
Immunoprecipitated TRIM25 and associated p53 and Mdm2 were detected by Western
blotting. Hybridization with antibodies directed against p300 and GAPDH were used as
nuclear (p300) and cytoplasmic (GAPDH) markers. (C) H1299 cells were co-transfected with
plasmids encoding Mdm2, V5-tagged hTRIM25 and p53 or with vector DNA for control. 24h
after transfection, cells were lysed. 5% of the cellular lysate were used to monitor expression
of p53, Mdm2 and TRIM25 by Western blotting (Input). From the remaining cellular lysate,
TRIM25 was immunoprecipitated using an antibody targeted against the V5-tag. Precipitation
with IgG was performed for control. The precipitates were resolved by gel electrophoresis.
Immunoprecipitated TRIM25 and associated p53 and Mdm2 were detected by Western
blotting. (D) H1299 cells were co-transfected with plasmids encoding p53, V5-tagged
hTRIM25 and Mdm2 or with vector DNA for control. 24h after transfection, cells were lysed
and processed as described in the legend to part A.
Figure S4: TRIM25 controls p53 activity. (A) HCT116 cells were transfected with a siRNA
directed against TRIM25 or with a control siRNA. 48h after transfection, cells were harvested
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and total RNA was prepared. The relative amounts of P21, 14-3-3σ, Mdm2, CDC25c and
TRIM25 mRNA were determined by qRT-PCR. Mean values and standard deviations were
calculated from the 2^dCT values of 3 independent experiments and blotted. Values of
mRNA in cells transfected with control siRNA were set to 1. P-values were calculated by the
two-tailed t-test (*P<0.05; n.s: not significant (B) HCT116 cells (HCTwt) and HCT116 cells
were p53 has been genetically removed (HCT116-/-) were transfected with a siRNA targeted
against TRIM25 or with a control siRNA. 48h after transfection, cells were analysed by
microscopy. C) HCT116 cells were transfected with two different TRIM25 siRNAs or with
control siRNA. 78h after transfection, cells were harvested and abundance of cleaved PARP,
cleaved Caspase 3, acetylated p53 and TRIM25 was monitored by Western blotting. (D) HCT
and HCT p53-/- cells were transfected in duplicates with a siRNA targeted against TRIM25 or
with a control siRNA. 48h after transfection, cells were analysed. I: MTT (1mg/ml f.c.) was
added 2 hours prior to lysis to one of the duplicates. Colour conversion of the tetrazolium salt
was monitored using an ELISA reader at 550 nm. Mean values and standard deviations
were calculated from three independent experiments and blotted. Values of cells transfected
with a control siRNA were set to 1. P-values were calculated by the two-tailed t-test (*P<0.05;
**P<0.01). II. Cells of the second duplicate were analysed by Western blotting for the
presence of p53, Mdm2 and TRIM25. (E) HCT116 (wt p53) cells were transfected in
duplicates with a siRNA targeted against TRIM25 or with a control siRNA. 48h after
transfection, cells were lysed. I: One of the duplicates was used to determine binding of p53
to consensus DNA. Mean values and standard deviations were calculated from three
independent experiments and blotted. Values of cells transfected with a control siRNA were
set to 1. II: Lysates of the second duplicate were analysed by Western blotting for the
presence of p53 and TRIM25. (F) MCF7 cells were transfected with two different TRIM25
siRNAs or with control siRNA in combination with either a control siRNA (to maintain the
amount of transfected siRNA constant) or with a siRNA targeting p300. 78h after
transfection, cells were harvested and abundance of P21, p300, TRIM25, acetylated p53 and
PCNA (for loading control) was monitored by Western blotting. The signals for P21,
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acetylated p53 and PCNA were quantified and the relative abundance of P21 and p53 was
calculated.
Figure S5: TRIM25 dampens the activation of p53 in response to DNA damage.
(A) MCF7 cells were transfected with a plasmid encoding TRIM25 or with vector DNA for
control. 24h after transfection, cells were treated with etoposide or with the vehicle for
control. 8h after treatment, cells were lysed and abundance of P21, p53, TRIM25 and PCNA
(for loading control) was monitored by Western blotting. The signals for P21 and PCNA were
quantified and the relative amount of P21 was calculated. (B) U2OS cells were transfected
with TRIM25 siRNA or a control siRNA. 40 h after transfection, cells were irradiated with rays (5 Gy), treated with etoposide (Eto: 50 µM f.c.) or left untreated for control (c). 8h after
DNA damage, cells were lysed. Abundances of TRIM25, P21, Mdm2 and Bax were
determined by Western blotting.
Figure S6: TRIM25 regulates p53 during medaka development. (A) Organisation of the
genomic locus of medaka TRIM25 on chromosome 1. The sequence of medaka TRIM25 has
been blasted onto the Ensemble (http://www.ensemble.org) database from where the data
have been exported. (B) Schematic drawing of the organisation of the two homologs of
TRIM25 of medaka. Both homologs possess a RING (really interesting new gene) domain,
two B-boxes (B1 and B2), and a coiled-coil domain. Homolog 1 has in addition a PRY
(domain associated with Spry domains) and a SPRY (domain present in SP1 and the
Ryanodine receptor) domain in its C-terminal part. (C) H1299 cells have been transfected
with plasmids encoding p53, Mdm2 and Myc-tagged ROR2, for control, together with
increasing amounts of plasmids encoding homolog 1 or homolog 2 of medaka TRIM25. 24h
after transfection, the amounts of p53, Mdm2, TRIM25 and ROR2 were determined by
Western blotting. Hybridisation with PCNA served for loading control. (D) Whole-mount in
situ hybridisation of one day-old medaka embryos incubated with digoxigenin-labeled RNA
riboprobes for p53, Mdm2 and TRIM25 homolog 1 and homolog 2 of medaka, respectively.
Shown is the dorsal view with the anterior part of the embryo showing to the left. (E)
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Embryos at 1, 2, 4 or 6 days after fertilisation were lysed in Trizol. Total RNA was extracted
and tested by RT-PCR for the expression of p53, Mdm2 and TRIM25 homolog 1 and
homolog 2 of medaka. RT-PCR for -Actin was used for internal control. (F) Morpholinoantisense oligonucleotides (MO) targeting both homologs of TRIM25 were injected into
fertilised medaka eggs. Control MOs at a concentration of 500 µM were injected for control.
One day after injection, the morphology of the embryos was assessed. The relative number
of affected embryos is displayed in the table (n: number of analysed embryos). (G)
Morpholino-antisense oligonucleotides (MO) targeting both homologs of TRIM25 in medaka
(concentration: 125 µM) were injected into fertilised medaka eggs either alone or together
with RNA from medaka TRIM25 (concentration: 300 ng/µl). One day after injection, the
morphology of the embryos was monitored by microscopy and graded into embryos with
normal or abnormal phenotype. Three independent experiments were performed and for
each experiment, the phenotype of between 23 and 29 embryos was determined. The graph
shows mean values and standard deviations of these experiments. The total number of
analysed embryos was set to 100%. P-values were calculated for embryos injected with
TRIM25 MOs and for embryos co-injected with TRIM25 MOs and mdTRIM25 mRNA by the
two tailed t-test (**P<0.01).
Figure S7: TRIM25 regulates p53 in mice and human tumours (A) Mice heterozygous for
p53 and TRIM25 were mated. The offsprings were genotyped and their weight was
determined. The graph shows the weight of sex matched individual mice of litter containing
p53/TRIM25 double knock-out mice at the age of 17 weeks (B) Tumour samples with known
(wt = wild type, mu = mutant, Stop = mutation resulting in a premature termination of
translation) and unknown (nd = not determined) p53 status were analysed for TRIM25 and
p53 expression by Western blotting. Hybridization with -Actin was performed for loading
control.
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