supplemental data statistics - genes &...

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


PMS2-/-




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+/+




PMS2-/-





Parameter: percentage of intestinal lesions (Figure 2b)


p-value

PMS2+/+



G3Terc-/- versus Terc+/+ <0.001

PMS2-/-




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)


p-value

PMS2+/+




PMS2-/-





Parameter: percentage of Ki67-positive cells (Figure 3b, right panel)


p-value

PMS2+/+




PMS2-/-





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+/+




PMS2-/-





Parameter: telomere length in intestinal sections (Figure 4b)

Statistical method: Wilcoxon-Mann-Whitney rank sum test

p-value

PMS2+/+



PMS2-/-



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)


p-value

PMS2+/+



PMS2-/-




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)


p-value

PMS2+/+




PMS2-/-




Siegl-Cachedenier et al. 10 Figure 7. A role for PMS2 in signaling telomeric dysfunction.

Parameter: percentage of p21-positive cells (Figure 7a)


p-value

PMS2+/+




PMS2-/-




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.


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.


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


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-/-


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-/-


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


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-/-


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-/-



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

PMS2-/-


PMS2+/+ PMS2-/-



Terc

+/+

G1T

erc-/-

G2T

erc-/-

G3T

erc-/-

PMS2+/+ PMS2-/-Q-FISH (MEFs)

a

b

0

100

200

300

400

500

0 20 40 60 80 100

Fre

qu

en

cy

Telomere length (kb)

0

100

200

300

400

500

0 20 40 60 80 100

Fre

qu

en

cy


0

100

200

300

400

500

0 20 40 60 80 100

Fre

qu

en

cy


0

100

200

300

400

500

0 20 40 60 80 100

Fre

qu

en

cy


0

100

200

300

400

500

0 20 40 60 80 100

Fre

qu

en

cy


0

100

200

300

400

500

0 20 40 60 80 100F

req

uen

cy


0

100

200

300

400

500

0 20 40 60 80 100

Fre

qu

en

cy


0

100

200

300

400

500

0 20 40 60 80 100

Fre

qu

en

cy


Number of telomeres = 3180 (2 mice)Mean length ± S.D. = 18.8±12.2 kbMedian length = 15.8 kbSignal free ends = 1.8% Telomeres <5 kb = 5.3%Telomeres >50 kb = 2.4%

Number of telomeres = 1580 (1 mouse)Mean length ± S.D. = 18.6±12.6 kbMedian length = 15.2 kbSignal free ends = 2.5% Telomeres <5 kb = 7.2%Telomeres >50 kb = 2.3%

10 kb

10 kb

10 kb

10 kb

10 kb

10 kb

10 kb

10 kb




Number of telomeres = 2867 (2 mice)Mean length ± S.D. = 12.6±12.7kbMedian length = 8.5 kbSignal free ends = 10.0% Telomeres <5 kb = 39.2%Telomeres >50 kb = 1.7%




Mr(K)

23.1

9.4

6.5

Terc

+/+

PMS2

+/+

Terc

+/+

PMS2

-/-

G1T

erc-

/- PM

S2+/

+

G1T

erc-

/- PM

S2-/-

G2T

erc-

/- PM

S2+/

+

G2T

erc-

/- PM

S2-/-

G3T

erc-

/- PM

S2+/

+

G3T

erc-

/- PM

S2-/-

3 33 3 3 33355 55 55 55 5555passage number:

TRF analysis


0

300

600

900

0 50 100 150 200

Fre

qu

en

cy


0

150

300

450

0 50 100 150 200

Fre

qu

en

cy


0

150

300

450

600

0 50 100 150 200

Fre

qu

en

cy


0

100

200

300

400

0 50 100 150 200

Fre

qu

en

cy


0

50

100

150

0 50 100 150 200

Fre

qu

en

cy


G3T

erc-/-

PM

S2+/

+ G

2Ter

c-/- P

MS2

+/+

#2344 Number of telomeres = 2328ML ± S.D. = 29.1 ± 28.1 kbMedian length = 20.2 kbSignal free ends = 15.5% Telomeres <5 kb = 16.7%Telomeres >50 kb = 21.0%

#2345 Number of telomeres = 2080ML ± S.D. = 33.1 ± 34.9 kbMedian length = 21.7 kbSignal free ends = 21.1% Telomeres <5 kb = 22.3%Telomeres >50 kb = 27.3%

#2182Number of telomeres = 2764ML ± S.D. = 40.5 ± 42.1kbMedian length = 23.5 kbSignal free ends = 19.9% Telomeres <5 kb = 20.6%Telomeres >50 kb = 34.7%

#2184Number of telomeres = 3092ML ± S.D. = 31.7 ± 34.0 kbMedian length = 19.0 kbSignal free ends = 27.0% Telomeres <5 kb = 27.3%Telomeres >50 kb = 26.5%

wild

type

#992 Number of telomeres = 2584ML ± S.D. = 51.9 ± 29.4 kbMedian length = 46.2 kbSignal free ends = 1.0%Telomeres <5 kb = 1.1%Telomeres >50 kb = 43.3%

G2T

erc-/-

PM

S2-/-

G3T

erc-/-

PM

S2-/-

0

20

40

60

80

0 10 20 30 40 50 60 70

passage number

accu

mu

late

d n

um

ber

of

po

pu

lati

on

do

ub

lin

gs (

PD

Ls)

0

20

40

60

0 10 20 30 40 50 60 70

passage number

accu

mu

late

d n

um

ber

of

po

pu

lati

on

do

ub

lin

gs (

PD

Ls)

0

20

40

60

80

0 10 20 30 40 50 60 70

passage number

accu

mu

late

d n

um

ber

of

po

pu

lati

on

do

ub

lin

gs (

PD

Ls)

0

20

40

60

0 10 20 30 40 50 60 70

passage number

accu

mu

late

d n

um

ber

of

po

pu

lati

on

do

ub

lin

gs (

PD

Ls)

0

10

20

30

0 10 20 30 40 50 60 70

passage number

accu

mu

late

d n

um

ber

of

po

pu

lati

on

do

ub

lin

gs (

PD

Ls)

Telomere length Immortalization

#992

#2344

#2182

#2184

#2345

a b

G2Terc-/- vs Terc+/+ p<0.001

G3Terc-/- vs Terc+/+ p<0.001

G2Terc-/-PMS2-/- vs Terc+/+ p<0.001

G3Terc-/-PMS2-/- vs Terc+/+ p<0.001


0

10

20

30

40

50

bre

aks a

nd

fra

gm

en

ts p

er

meta

ph

ase (

%)

2/50

5/51(p=0.251)

13/51(p=0.002)

18/50(p<0.001)

7/50

8/50(p=0.779) 7/51

(p=0.968)

8/50(p=0.779)

PMS2+/+

PMS2–/–

0

20

40

60

80

en

d-t

o-e

nd

fu

sio

ns p

er

meta

ph

ase (

%)

0/50

5/50(p=0.005)

5/52(p=0.006)

31/50(p<0.001)

4/50

6/50(p=0.500)

11/51(p=0.055)

23/50(p<0.001)

PMS2+/+

PMS2–/–

0

10

20

30

40

sig

na

l-fr

ee

en

ds

pe

r c

hro

mo

so

me

(%

)

23/2601

91/2535(p<0.001)

PMS2+/+

PMS2–/–

133/2751(p<0.001)

318/2428(p<0.001)

2/2388

86/2279(p<0.001)

346/3014(p<0.001)

734/2655(p<0.001)

signal-free ends

Terc+/+ G1Terc-/- G2Terc-/- G3Terc-/- Terc+/+ G1Terc-/- G2Terc-/- G3Terc-/-

breaks and fragments

a

end-to-end fusions



Terc+/+ p<0.001 G1Terc-/- p=0.735G2Terc-/- p<0.001G3Terc-/- p<0.001

PMS2+/+ vs PMS2-/-

comparison between genotypes

Terc+/+ p=0.013 G1Terc-/- p=0.749G2Terc-/- p=0.094G3Terc-/- p=0.108

PMS2+/+ vs PMS2-/-



PMS2+/+ vs PMS2-/-


Siegl-Cachedenier_Supplementary Figure 7a

0

2

4

6

8

oth

er

ab

err

ati

on

s p

er

me

tap

ha

se

(%

)

0/500/51(p=1)

0/52(p=1)

0/50(p=1)

3/50

2/50(p=0.646)

1/51(p=0.298)

1/50(p=0.307

PMS2+/+

PMS2–/–

0

2

4

6

8

10

perc

en

tag

e p

er

meta

ph

ase

0/500/51(p=1)

1/52(p=0.227)

4/50(p=0.013)

1/50

0/50(p=0.219)

0/51(p=0.215)

0/50(p=0.219)

PMS2+/+

PMS2–/–

0

5

10

15

20

25

pe

rce

nta

ge

pe

r m

eta

ph

as

e

0/500/51(p=1)

1/52(p=0.227)

3/50(p=0.032)

9/50

2/50(p=0.025)

0/51(p<0.001)

2/50(p=0.025)

PMS2+/+

PMS2–/–

bivalent recombination figures and chromatid crosslinks

complex aberrations and fusions


minichromosomes

b



Terc+/+ p=0.223 G1Terc-/- p=1G2Terc-/- p=0.223G3Terc-/- p=0.013

PMS2+/+ vs PMS2-/-


Terc+/+ p<0.001 G1Terc-/- p=0.079G2Terc-/- p=0.223G3Terc-/- p=0.646

PMS2+/+ vs PMS2-/-



PMS2+/+ vs PMS2-/-


Siegl-Cachedenier_Supplementary Figure 7b

wild ty

pe

G1Terc

-/-

G1Terc

-/- PMS2-

/-

PMS2-/-

G2Terc

-/-

G2Terc

-/- PMS2-

/-

G3Terc

-/-

G3Terc

-/- PMS2-

/-

100 bp

water

D6Mit59

wild ty

pe

G1Terc

-/-

G1Terc

-/- PMS2-

/-

PMS2-/-

G2Terc

-/-

G2Terc

-/- PMS2-

/-

G3Terc

-/-

G3Terc

-/- PMS2-

/-

100 bp

200 bp

water

D1Mit62

36.36 % 71.43 % 44.44 % 85.71 % 62.50 % 44.44 % 100 % 100 %

200 bp


b

a

c

β-actin

p21

IR: – 8h 18h – 8h 18h – 8h 18h – 8h 18h10Gy 10Gy 10Gy 10Gy

wt PMS2-/- G2Terc-/- G2Terc-/-

PMS2-/-

0

2

4

6

8

pe

rce

nta

ge

of

p5

3 p

os

itiv

e c

ell

s

15

/22

04

4/1

53

8

p=0.074

11

3/1

88

2

57

/20

09

p<0.001

p53 expression

G2Terc-/-Terc+/+

PMS2 +/+

PMS2 –/–

PMS2+/+

Terc+/+ vs G2Terc-/- p<0.001 p<0.001PMS2-/-


0

20

40

60

80

100

120

140

160

180

wt PMS2-/- G2 Terc-/- G2 Terc-/- PMS2 -/-

n=3n=6

n=6

n=4

p=0.032

rela

tive

p21

leve

ls v

s w

tre

lativ

e p2

1 le

vels

vs

wt

wt G2Terc-/- G2Terc-/-PMS2-/-

PMS2-/-

p=0.027p=0.62

p=0.54

Siegl-Cachedenier_Supplementary Figure 9a-c

supplemental data statistics - genes &...

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