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Siegl-Cachedenier et al. 1
SUPPLEMENTAL DATA
STATISTICS
Statistical comparisons between different generations of Terc-/- mice
Figure 1. PMS2-deficiency rescues organismal survival of mice with dysfunctional telomeres.
Parameter: survival curves (Kaplan Meyer plot)
Statistical method: logrank test
p-value
PMS2+/+
G1Terc-/- versus Terc+/+ 0,003
G2Terc-/- versus Terc+/+ <0,001
G3Terc-/- versus Terc+/+ <0,001
PMS2-/-
G1Terc-/- versus Terc+/+ 0,030
G2Terc-/- versus Terc+/+ 0,017
G3Terc-/- versus Terc+/+ 0,203
Siegl-Cachedenier et al. 2 Figure 2. Rescue of malignant tumors and degenerative pathologies in Terc-/-/PMS2-/- mice.
Parameter: percentage of malignant tumors (Figure 2a)
Statistical method: Chi squares test
p-value
PMS2+/+
G1Terc-/- versus Terc+/+ 0,111
G2Terc-/- versus Terc+/+ 0,028
G3Terc-/- versus Terc+/+ 0,141
PMS2-/-
G1Terc-/- versus Terc+/+ 0,186
G2Terc-/- versus Terc+/+ 0,036
G3Terc-/- versus Terc+/+ 0,021
Siegl-Cachedenier et al. 3
Parameter: percentage of intestinal lesions (Figure 2b)
Statistical method: Chi squares test
p-value
PMS2+/+
G1Terc-/- versus Terc+/+ <0,001
G2Terc-/- versus Terc+/+ <0,001
G3Terc-/- versus Terc+/+ <0.001
PMS2-/-
G1Terc-/- versus Terc+/+ 0,006
G2Terc-/- versus Terc+/+ 0,110
G3Terc-/- versus Terc+/+ 0,077
Siegl-Cachedenier et al. 4 Figure 3. Rescue of proliferation defects but not apoptosis in Terc-/-/PMS2-/- mice
Parameter: percentage of caspase 3-positive cells (Figure 3b, left panel)
Statistical method: Chi squares test
p-value
PMS2+/+
G1Terc-/- versus Terc+/+ <0,001
G2Terc-/- versus Terc+/+ 0,003
G3Terc-/- versus Terc+/+ <0.001
PMS2-/-
G1Terc-/- versus Terc+/+ <0,001
G2Terc-/- versus Terc+/+ <0,001
G3Terc-/- versus Terc+/+ <0,001
Siegl-Cachedenier et al. 5
Parameter: percentage of Ki67-positive cells (Figure 3b, right panel)
Statistical method: Chi squares test
p-value
PMS2+/+
G1Terc-/- versus Terc+/+ <0,001
G2Terc-/- versus Terc+/+ <0,001
G3Terc-/- versus Terc+/+ <0.001
PMS2-/-
G1Terc-/- versus Terc+/+ <0,001
G2Terc-/- versus Terc+/+ <0,001
G3Terc-/- versus Terc+/+ <0,001
Siegl-Cachedenier et al. 6
Figure 4. PMS2-deficiency does not rescue telomere shortening in telomerase deficient mice
Parameter: telomere length in MEFs (Figure 4a)
Statistical method: Wilcoxon-Mann-Whitney rank sum test
p-value
PMS2+/+
G1Terc-/- versus Terc+/+ 0,030
G2Terc-/- versus Terc+/+ <0.001
G3Terc-/- versus Terc+/+ <0.001
PMS2-/-
G1Terc-/- versus Terc+/+ <0.001
G2Terc-/- versus Terc+/+ <0,001
G3Terc-/- versus Terc+/+ <0,001
Siegl-Cachedenier et al. 7
Parameter: telomere length in intestinal sections (Figure 4b)
Statistical method: Wilcoxon-Mann-Whitney rank sum test
p-value
PMS2+/+
G1Terc-/- versus Terc+/+ <0,001
G2Terc-/- versus Terc+/+ <0,001
PMS2-/-
G1Terc-/- versus Terc+/+ <0,001
G2Terc-/- versus Terc+/+ <0,001
Siegl-Cachedenier et al. 8 Figure 5. Similar numbers of γ-H2AX-positive cells in Terc-/-/PMS2+/+ and Terc-/-/PMS2-/- small intestine sections
Parameter: percentage of γ-H2AX-positive cells (Figure 5a)
Statistical method: Chi squares test
p-value
PMS2+/+
G1Terc-/- versus Terc+/+ <0,001
G2Terc-/- versus Terc+/+ <0,001
PMS2-/-
G1Terc-/- versus Terc+/+ <0,001
G2Terc-/- versus Terc+/+ <0,001
Siegl-Cachedenier et al. 9
Figure 6. Telomere recombination in Terc-/-/PMS2-/- MEF is equivalent to that of single mutant Terc-/- MEF
Parameter: percentage of sister chromatid exchange (Figure 6a)
Statistical method: Chi squares test
p-value
PMS2+/+
G1Terc-/- versus Terc+/+ <0,001
G2Terc-/- versus Terc+/+ <0,001
G3Terc-/- versus Terc+/+ <0.001
PMS2-/-
G1Terc-/- versus Terc+/+ <0,001
G2Terc-/- versus Terc+/+ 0,017
G3Terc-/- versus Terc+/+ <0,001
Siegl-Cachedenier et al. 10 Figure 7. A role for PMS2 in signaling telomeric dysfunction.
Parameter: percentage of p21-positive cells (Figure 7a)
Statistical method: Chi squares test
p-value
PMS2+/+
G1Terc-/- versus Terc+/+ <0,001
G2Terc-/- versus Terc+/+ <0,001
G3Terc-/- versus Terc+/+ <0.001
PMS2-/-
G1Terc-/- versus Terc+/+ <0,001
G2Terc-/- versus Terc+/+ <0,001
G3Terc-/- versus Terc+/+ <0,001
Siegl-Cachedenier et al.
Supplementary Figures
Supplementary Figure 1. PMS2-deficiency rescues organismal survival of mice
with dysfunctional telomeres. a. Median survival obtained from Kaplan-Meyer plots
of mice of the indicated genotypes. Statistical comparisons using the log rank test are
also shown. b. Mean lifespan of mice of the indicated genotypes. Statistical
comparisons using the Student´s t-test are also shown. Comparisons between
different mouse generations are also shown in the tables below the graphs.
Supplementary Figure 2. Degenerative pathologies in Terc-/-/PMS2-/- mice.
Percentage of mice of the indicated genotype showing degenerative patologies in the
indicated organs at the time of death. Note that intestinal lesions are the most
abundant pathology in these mice. Statistical comparisons using the Chi-square test
are also shown. Comparisons between different mouse generations are also shown
in the table.
Supplementary Figure 3. PMS2-deficiency does not rescue critically short
telomeres in telomerase deficient mice. Percentage of telomeres shorter than 5kb
(<5kb) as determined by Q-FISH in primary MEF (passage 2) of the indicated
genotypes (see also Fig. 4a). Statistical comparisons using the Chi squared test are
shown. Comparisons between different mouse generations are also shown in the
table.
Siegl-Cachedenier et al. 2
Supplementary Figure 4. PMS2-deficiency does not rescue telomere shortening
in telomerase deficient mice. a. Telomere fluorescence as determined by Q-FISH
in 1-to-2 primary MEF (passage 2) of the indicated genotypes. More than 1,500
telomeres were analyzed per genotype by Q-FISH. Mean telomere length in kb and
standard error are shown. Statistical significance using the Wilcoxon-Mann-Whitney
rank sum test is indicated. b. Percentage of telomeres shorter than 5kb (<5kb) as
determined by Q-FISH in primary MEF (passage 2) of the indicated genotypes.
Statistical comparisons using the Chi squared test are shown. Comparisons between
different mouse generations are also shown in the table.
Supplementary Figure 5. Telomere length in Terc-/-/PMS2-/- MEF. Telomere length
as determined by TRF in MEF of the indicated genotypes. No clear differences in
TRF fragment size were detected between single Terc-/- MEF and the corresponding
double mutant Terc-/-/PMS2-/- controls.
Supplementary Figure 6. PMS2-deficiency rescues in vitro proliferation defects
in Terc-/-/PMS2-/- MEF. a. Telomere length as determined by Q-FISH in immortalized
MEF at passage 35 of the indicated genotypes. Numbers identify individual MEF
cultures. More than 2,000 telomeres were analyzed per genotype by Q-FISH. Mean
telomere length in kb and standard error are shown. Statistical significance using the
Wilcoxon-Mann-Whitney rank sum test is indicated It is important to note that
telomere length values shown in this experiment can be compared between
genotypes as Q-FISH was performed on the same day and in parallel. These values,
however, cannot be directly compared with those shown in Fig. 4a as they
correspond to an independent Q-FISH experiment with primary (passage 2) MEF. b.
Siegl-Cachedenier et al. 3
Accumulated number population doublings with increasing passage (3T3 passaging
protocol; see Materials and Methods) in MEF of the indicated genotypes. The arrows
indicate the passage number at which the MEF culture escapes the so-called
“premature senescence” arrest (see Materials and Methods). Note that late
generation G3 Terc-/- MEF need more than 50 passages to escape premature
senescence, while this is achieved with only 15 passages in the case of double
mutant G3 Terc-/-/PMS2-/- MEF.
Supplementary Figure 7. Severe telomere dysfunction and chromosomal
instability in Terc-/-/PMS2-/- MEF. a,b. Quantification of frequency of the indicated
chromosomal aberrations per chromosome or per metaphase in primary MEF
(passage 3) of the indicated genotypes. Statistical calculations using the Chi-square
test are shown. Comparisons between genotypes are also shown in the table. The
total number of chromosomes or metaphases scored for the analysis are indicated on
top of each bar. Representative examples of the indicated complex chromosomal
aberrations are shown in part “b”. Note that both signal free ends and end-to-end
fusions are further increased in Terc-/-/PMS2-/- MEF compared to the single mutant
controls.
Supplementary Figure 8. Microsatellite instability Terc-/-/PMS2-/- MEF. Total DNA
from the tail of mice of the indicated genotype was used for the microsatellite
instability analysis by PCR amplification (Materials and Methods). The D1Mit62 CA
repeat loci was used as a negative control, as this locus is stable. The frequency of
appearance of novel length alleles of the D6Mit59 locus is indicated for each
genotype in white bold characters. Note that microsatellite instability was similar or
Siegl-Cachedenier et al. 4
increased in Terc-/-/PMS2-/- mice compared to the single mutant PMS2-deficient
controls.
Supplementary Figure 9. A role for PMS2 in signaling telomeric dysfunction. a.
Western blot analysis of p21 protein levels in primary MEF (passage 2) of the
indicated genotypes before (-) and after (8h and 18h) ionizing radiation (positive
control for p21 accumulation). Actin was used as a loading control. b. Quantification
of p21 protein levels in 3-to-6 independent MEF cultures per genotype before (-) and
after (8h and 18h) ionizing radiation after normalization to actin levels. Note a
statistically significant decreased p21 accumulation in Terc-/-/PMS2-/- MEF compared
to the single mutant controls. c. Percentage of cells of the indicated genotype
showing p53-positive staining at GI tract. The total number of cells counted for the
analysis is also indicated.
0
20
40
60
80
100
120
mean
lif
esp
an
(w
eeks)
n=16
n=15
n=39
n=20
n=46
n=34
n=4
n=6
p<0.001
p=0.424
p<0.001
p=0.534
0
20
40
60
80
100
120
me
dia
n s
urv
iva
l (w
ee
ks
)
p<0.001
p=0.841
p<0.001
p=0.226
n=29
n=23
n=47
n=33
n=55
n=46
n=5
n=6
PMS2 +/+
PMS2 –/–
PMS2 +/+
PMS2 –/–
G2Terc-/- G3Terc-/-Terc+/+ G1Terc-/-
G2Terc-/- G3Terc-/-Terc+/+ G1Terc-/-
median survival(from Kaplan Meyer plots)
mean lifespan(only valid deaths)
Siegl-Cachedenier_Supplementary Figure 1a,b
a
b
PMS2+/+
Terc+/+ vs G1Terc-/- p<0.001 p=0.029Terc+/+ vs G2Terc-/- p<0.001 p=0.008Terc+/+ vs G3Terc-/- p<0.001 p=0.839
PMS2-/-
comparison between generations
PMS2+/+
Terc+/+ vs G1Terc-/- p=0.003 p=0.030Terc+/+ vs G2Terc-/- p<0.001 p=0.017Terc+/+ vs G3Terc-/- p<0.001 p=0.203
PMS2-/-
comparison between generations
degenerative pathologies
PMS2+/+
Terc+/+ vs G1Terc-/- p=0.153 p=0.063Terc+/+ vs G2Terc-/- p=0.634 p=0.287Terc+/+ vs G3Terc-/- p=0.798 p=0.144
PMS2-/-
comparison between generations
0
50
100
150
200
250
300
mic
e a
ffe
cte
d (
%)
54.6
n=11
n=26
n=17
n=28
n=19
27.3
72.7
22.2
44.4
100.0
100.025.0
85.7
41.2
15.8
30.8
84.6 29.4
35.35.9
17.7
10.7
3.6
7.2
21.1
47.3
21.1
5.3
25.0
75.0
50.0
75.0
71.4
11.1
9.1
27.3
27.311.1 23.1
46.1 11.829.4 3.657.4
21.1
25.0
p=0.010
p=0.004
p=0.002
n=9
p=0.763
n=4
n=4
kidneyspleenliver
intestine heart and lungreproductive tractothers
PMS2+/+ PMS2-/-
Terc+/+ G1Terc-/- G2Terc-/- G3Terc-/-
PMS2+/+ PMS2-/- PMS2+/+ PMS2-/- PMS2+/+ PMS2-/-
Siegl-Cachedenier_Supplementary Figure 2
Siegl-Cachedenier_Supplementary Figure 3
PMS2+/+
Terc+/+ vs G1Terc-/- p<0.001 p<0.001Terc+/+ vs G2Terc-/- p<0.001 p<0.001Terc+/+ vs G3Terc-/- p<0.001 p<0.001
PMS2-/-
comparison between generations
0
10
20
30
40
perc
en
tag
e o
f te
lom
ere
s <
5kb
90/1584114/1580
p=0.079
154/1680
227/1580
p<0.001 352/1596
523/1575
p<0.001
452/1552
496/1572
p=0.140
PMS2+/+ PMS2-/-
Terc+/+ G1Terc-/- G2Terc-/- G3Terc-/-
PMS2+/+ PMS2-/- PMS2+/+ PMS2-/- PMS2+/+ PMS2-/-
0
10
20
30
40
pe
rce
nta
ge
of
telo
me
res
<5
kb
170/3180114/1580
p=0.010
154/1680
227/1580
p<0.001
801/3180
838/2867
p<0.001
904/3104 933/3232
p=0.822
PMS2+/+
Terc+/+ vs G1Terc-/- p<0.001 p<0.001Terc+/+ vs G2Terc-/- p<0.001 p<0.001Terc+/+ vs G3Terc-/- p<0.001 p<0.001