Alignment of ERV9-LTR sequences of human genes.

Blast search identified candidate genes that contain ERV9 LTRs. The alignment shows the

LTR sequences of known human genes with the consensus sequence indicated below.

Consensus

AACCTGCTCAGGTCCCCTTCCATGCTGTGGAAGCTTTGTTTTTTTGCACTTT-GCAGTAAAATCTGTTCAGGTCGTTTTCCATAGTGTGGAAGCTTTGTTCTTTCGC-TCTTTGCAATAA ----CACTGAGGTCACCCTCCAGGCTGTGGAACCTTTGTTCTTTCACTCTTT-GCAATAA ----------------CTTCAACACTGTGGAAGCTTTGTTCTTTCGCTCTTTTGCAATAA AACTTGCTGGAGTCGCCTTCTGTGCTGTGGAAGTTTTGTTGTTTTGCTTGTTTGCAATAA ** . . ******* ****** *** .* ** ***.*** 

ATCTTGCTGCTGCTCATTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCG- ATCTTGCTGCTGCTCACTCTTTGGGTTCACACTGCCTTTATGAGCTGTAACACTCACCAC ATCTTGCTGCTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACTGG ATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCGC CCGTTGCTGCTGCTTACTGTTTGGGTCCGCACTGCCTTTATGAGCTGTAACGCTCACCGT. *****.***** * * ******* *.**********************.***** .

TGAAGGTCTGCAGCATCACTCCTGAAACCTGTGAAACAACGAACCCCCCGGGGAGAAACGGA-AGGTCTACAGCTTTACTCCTGAAGCCAGCGAGATCACGAACCCA-CTGGGAGAAACG GA-ATGTCTGCAGCTTCACTCCTGAAGCCAGCGAGACCACGAACCCA-CCAGGAGGAACA GAAAGGTCTGCAGCTTCACTCCTGAAGCCAGCGAGCCCACGAGCCCA-CAGGGAGGAATG GA-AGGTCTGCAGCTTCGTTCTTAAAGCCAGCCACATCACGGACCCA-CCGGGAGAAACG . * ****.****:* . ** *.**.**:* * . .***..***. * .****.** . 

AACAACTCCAGACTCGCCGCCTGAAGAGCTGTAACTCTCACTTCGAAGGTCTGCAGCTTC AACAACTCCAGACGCACTACCTTAAGAGCTGTAACACTCACTGCGAAGGTCCGCAGCTGC AACAACTCCAGACGCGCAGCCTTAAGAGCTGTAACACTCACCGCGAAGGTCTGCAGCTTCAACAACTCCAGATGCACCGCCTTAAGAGCTGTAACACTCACGGCGAAGGTCTGCAGCTTCAATAACTCCAAACACGCCACCTTAAGAGCCGTAGCACTCATGATGAAGGTCCGTAGCTTC ** *******.* *.* .*** ****** ***.*:**** ******* * **** *

GCTCCTGAG-TCAGTGAAACCACGAACCCACCGGAAGGAAGAAACTCTGAACACATCCAAATTCCTGAG-CCAGCGAGACCACAAACCCACCAGAAGGAAGAAACTTCGAACGCATCCGA ACTCCTGAG-CCAGCCAGACCACGAACCCACCAGAAGGAAGAAACTCCAAACACATCCGA ACTCCTGAG-CCAGCGAGACCAGGAACCCACCAGAAGGAAGAAACTCCGAACACATCCGA ACTCCTGAAGTCAGCGAGATCACGAATCTGCCAGAAGGAAGAAACTCCGAACACATGCGA . ******. *** *.* ** .** * .**.************* .***.*** .*

ACATCAGAAGGAACAAACTCCGGACACGCAGCCTTTAAGAATTGTAACACTCA-CCGCGA ACATCAGAAGGAACAAACTCCAGACACGCCGCCTTTAGGAACTGTAACACTCAACCGCGA ACATCAGAAGGAGCAAACTCCTGACACGCCACCTTTAAGAACCGTGACACTCA-ACGCTA ACATCAGAAGGAACAAACTCCAGACGGCGCCACCTTAAGAGCTGTAACACTCA-CCGCCA GCATCAGAAGAAACAAACTTGGGACACGCTGCCTTTAAAAACTGTAACACTCA-CCGCCA.*********.*.****** ***. ..* ****.*. **.******* .*** * 

GGGTCCGTGGCTTCATTCTTGAAGTCAGTGAGACGAAGAACCCACCAATTCCGGTAGGGGGTCCGCGGCTTCATTCTTGAAGTCAGTGAGACCAAGAACCCACCAATTCCGGACACGGGTCCGCGGCTTCATTCTTGAAGTCAGTGAGACCAAGAACCCACCAATTCCGGACACGGGTCCAGGGCTTCGTTCTTGAAGTCAGTGAGACCAAGAACCCACCAATTCCGGACGCGGGTCCGCGGCTTCGTTCTTGAAGTCAGTGAGACCAAGAATCCGCCGATTCTGGACAC******. ******.******************* ***** **.**.**** **:..

IRGM

CGREF1GBP5KCNN2PGPEP1LConsensus

IRGMCGREF1GBP5KCNN2PGPEP1LConsensus

IRGMCGREF1

GBP5KCNN2PGPEP1LConsensus

IRGMCGREF1GBP5KCNN2PGPEP1LConsensus

IRGMCGREF1GBP5KCNN2

PGPEP1LConsensus

IRGMCGREF1GBP5KCNN2PGPEP1L

Consensus

IRGMCGREF1GBP5KCNN2

PGPEP1L

297296292282298

356356351

341357

238237233223238

414

414

409399

415

178

177173163

178

118

119115104

120

59

5955

4460

Figure S1

testis

1031 bp -

2000 bp -

1500 bp -

500 bp -

3000 bp -

+ - + - + - + - + - + - + - + - RT

GH+TSA

700 bp -

testis GH+TSA testis GH+TSA testis GH+TSA

RACE_for1 RACE_for2 RACE_for3 RACE_for4

Primer Mix

3’RACE PCR products.

Pools of PCR-products obtained by the 3’RACE protocol using 4 LTR-specific forward

oligonucleotides and the reverse Universal Primer Mix, visualized by agarose gel

electrophoresis and ethidium bromide staining. Similar products were obtained using the 4

degenerated forward oligonucleotides.

Figure S2

ex1_revLTR +339_for

CDSex1LTR

LTR +1006_for ex1 mid_rev

CDSex1LTR

TNFRSF10B LTR-transcript 1: 4413 bp (splicing onto splice acceptor site within exon 1)

TNFRSF10B LTR-transcript 2: 4664 bp (splicing onto sequence upstream of exon 1)

AACACTGTGGAAGCTTCCTTCTTTCCCTCTGCAATAAATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCTTGAAGGTCTGCAGCTTCACTCTTGAAGCCAGCGAGACCACGAGCCCACCAGGAGGAAAGAACAACTCCAGACCCACTGCCTTAAGAGCTGTAACACTCACTGGGAAGGTCTGCAGCTTCACTCCTGAGCCAGTGAGACCACGAACCCACCAGAAGGAAGAAACTCCGAACACATCCGAACATCAGAAGGAACAAACTCCAGACACGCCGCCTTTAAGAACTGTAACACTCACCGCGAGGGTCCGAGGCTTCATTCTTGAAGGCAGTGAGACCAAGAACCCACCAATTCCGGACACAGTACCATGAAGGAATGAAAATACATAACAATTTGCACATTGGATCTGATTCGCCCCGCCCCGAATGACGCCTGCCCGGAGGCAGTGAAAGTACAGCCGCGCCGCCCCAAGTCAGCCTGGACACATAAATCAGCACGCGGCCGGAGAACCCCGCAATCTCTGCGCCCACAAAATACACCGACGATGCCCGATCTACTTTAAGGGCTGAAACCCACGGGCCTGAGAGACTATAAGAGCGTTCCCTACCGCCATGGAACAACGGGGACAGAACGCCCCGGCCGCTTCGGGGGCCCGGAAAAGGCACGGCCCAGGACCCAGGGAGGCGC […] TATTTATGAATCCATGACCAAATTAAATATGAAACCTTATATAAAAA

AACACTGTGGAAGCTTCCTTCTTTCCCTCTGCAATAAATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCTTGAAGGTCTGCAGCTTCACTCTTGAAGCCAGCGAGACCACGAGCCCACCGGGAGGAAAGAGAAAGAGAGAAAGGAAGGAAAGAGAAAGCAGGAAGGACGGAAAGAAGACGAAAGAACGAAAGAAAACGAAAGAAAAAAGGAAAGAAGAGAGAAGGAGAGAACAGAAGGGGCAGGTGCCCCTGGGAAGGGGAGAAGATCAAGACGCGCCTGGAAAGCGGACTCTGAACCTCAAGACCCTGTTCACAGCCAAGCGCGCGACCCCGGGAGGCGTCAACTCCCCAAGTGCCTCCCTCAACTCATTTCCCCCAAGTTTCGGTGCCTGTCCTGGCGCGGACAGGACCCAGAAACAAACCACAGCCCGGGGCGCAGCCGCCAGGGCGAAGGTTAGTTCCGGTCCCTTCCCCTCCCCTCCCCACTTGGACGCGCTTGCGGAGGATTGCGTTGACGAGACTCTTATTTATTGTCACCAACCTGTGGTGGAATTTGCAGTTGCACATTGGATCTGATTCGCCCCGCCCCGAATGACGCCTGCCCGGAGGCAGTGAAAGTACAGCCGCGCCGCCCCAAGTCAGCCTGGACACATAAATCAGCACGCGGCCGGAGAACCCCGCAATCTCTGCGCCCACAAAATACACCGACGATGCCCGATCTACTTTAAGGGCTGAAACCCACGGGCCTGAGAGACTATAAGAGCGTTCCCTACCGCCATGGAACAACGGGGACAGAACGCCCCGGCCGCTTCGGGGGCCCGGAAAAGG […] TATTTATGAATCCATGACCAAATTAAATATGAAACCTTATATAAAAA

LTR +41_for upst ex1_rev

CDSex1LTR

TNFRSF10B LTR-transcript 3: 4532 bp (splicing onto sequence upstream of exon 1)

AACACTGTGGAAGCTTCCTTCTTTCCCTCTGCAATAAATCTTGCTACTGCTCACTCTTTGGGTCCACACTGCCTTTATGAGCTGTAACACTCACCTTGAAGGTCTGCAGCTTCACTCTTGAAGCCAGCGAGACCACGAGCCCACCAGGAGGAAAGAACAACTCCAGACCCACTGCCTTAAGAGCTGTAACACTCACTGGGAAGGTCTGCAGCTTCACTCCTGAGCCAGTGAGACCACGAACCCACCAGAAGGAAGAAACTCCGAACACATCCGAACATCAGAAGGAACAAACTCCGGACAGGACCCAGAAACAAACCACAGCCCGGGGCGCAGCCGCCAGGGCGAAGGTTAGTTCCGGTCCCTTCCCCTCCCCTCCCCACTTGGACGCGCTTGCGGAGGATTGCGTTGACGAGACTCTTATTTATTGTCACCAACCTGTGGTGGAATTTGCAGTTGCACATTGGATCTGATTCGCCCCGCCCCGAATGACGCCTGCCCGGAGGCAGTGAAAGTACAGCCGCGCCGCCCCAAGTCAGCCTGGACACATAAATCAGCACGCGGCCGGAGAACCCCGCAATCTCTGCGCCCACAAAATACACCGACGATGCCCGATCTACTTTAAGGGCTGAAACCCACGGGCCTGAGAGACTATAAGAGCGTTCCCTACCGCCATGGAACAACGGGGACAGAACGCCCCGGCCGCTTCGGGGGCCCGGAAAAGG […] TATTTATGAATCCATGACCAAATTAAATATGAAACCTTATATAAAAA

Novel TNFRSF10B LTR-transcripts .

Sizes and sequences of the newly identified TNFRSF10B transcripts expressed from the

upstream ERV9-LTR are shown. Alternative splicing results in three novel transcripts that are

predicted to encode the same TNFRSF10B protein, due to use of the same start codon.

Binding sites for the oligonucleotides used for specific transcript amplification are indicated by

small arrows. The ERV9-LTR-sequences are marked in grey, and exon 1 of TNFRSF10B is

marked in red.

Figure S3

[...]

A

Figure S4

B

Insertion of the ERV9-LTR-sequence upstream of TNFRSF10B in primates.

A. Alignment of genomic sequences of diverse primate species including hominids containing

the upstream region of each TNFRSF10B gene, using the ClustalX algorithm. The ERV9-LTR

(yellow shading) is only inserted from Homo sapiens to Hylobatidae.

B. Sequence comparison between ERV9 subfamily sequences by the ClustalX algorithm

reveals that the TNFRFS10B-associated ERV9 LTR belongs to subfamily IX of ERV9 [47]. The

radial alignment tree was generated using FigTree v1.4.0

(http://tree.bio.ed.ac.uk/software/figtree/).

Figure S4, continued

A

F

NCCIT cells

DMSO 50ng/ml TRAIL 0.5µM TSA TRAIL + TSA SSC2 DR5_A DR5_B SSC2 DR5_A DR5_B SSC2 DR5_A DR5_B SSC2 DR5_A DR5_B

MW (kDa)

PARP1cleaved PARP 100

Beta-Actin40

35 Caspase-3

cleavedCaspase-315

E

1 E-01

1 E-02

1 E-03

TSA [µM]

TN

FR

SF

10

Bto

tal v

s.

RP

LP

0 m

RN

A le

vel

(lo

gari

thm

ic s

cale

)

**

B

TN

FR

SF

10

A

vs.

RP

LP

0 m

RN

A

(lin

ear

scal

e) 0.0015

0.0010

0.0005

0.0000

TSA [µM]

C D0.006

0.004

0.002

0.000TN

FR

SF

10

B v

s.

RP

LP

0 m

RN

A

(lin

ear

scal

e)

DR5_BDR5_A

SSC2

siRNA

Ta

rge

t vs

. RP

LP0

mR

NA

(lin

ear

sca

le)

DMSO 2µM 5µM 10µM

Nutlin 3a [24h]

TNFRSF10B total

CDKN1A

**

TNFRSF10B LTR transcript 2

1 E-04

Figure S5Susa cells 1618-K cells

D 0.5 1 2

1 E-01

1 E-02

1 E-03

1 E-04

D 0.5 1 2 1 5

TSA [µM] SAHA [µM]

*

**

***

D 0.5 1 2 1 5

TSA [µM] SAHA [µM]

1 E-01

1 E-02

1 E-03

1 E-04

**

0.06

0.04

0.02

0.00

0.08

**

*

*

**TNFRSF10A (DR4)

D 0.5 1 2

day 0 1 2 3 4 5 0 1 2 3 4 50 1 2 3 4 5 0 1 2 3 4 5

Cel

l co

nflu

en

cy [

%]

8060

40200

8060

40

200

100

100

SSC2 DR5_A DR5_B

DMSO 50ng/ml TRAIL [10h] 0.5µM TSA + 50ng/ml TRAIL0.5µM TSA [12h]

A. TSA treatment also increased the expression of total TNFRSF10B in NCCIT, Susa and

1618-K testicular cancer cells, as determined by qRT-PCR (mean values of three

independent experiments). p-values were calculated using Student’s t-test (ns: p>0.05, *:

p<0.05, **: p<0.01, ***: p<0.001).

B. Expression of TNFRSF10A mRNA is not induced upon treatment of GH cells with TSA, as

determined by qRT-PCR (mean values of three independent experiments).

C. The MDM2 inhibitor Nutlin 3a does not activate transcription of TNFRSF10B from the ERV9-

LTR-promoter. Total mRNAs isolated from GH cells treated with increasing concentrations of

Nutlin 3a or the solvent DMSO for 24 hrs were subjected to quantitative real-time-RT-PCR.

Expression levels of total TNFRSF10B, TNFRSF10B LTR-transcript 2, or CDKN1A were

normalized to RPLP0 (mean values of three independent experiments). Transcription of

CDKN1A was induced as a consequence of p53 activation.

D. Knockdown of TNFRSF10B expression by specific siRNAs. GH cells were transfected with

two different siRNAs against the TNFRSF10B gene product DR5 (DR5_A and DR5_B) or a

control scrambled SSC2 siRNA and harvested after 36 hrs for quantification of TNFRSF10B

mRNA levels by real-time RT-PCR. RPLP0 served as a reference gene.

E. siRNA-mediated depletion of TNFRSF10B expression in GH cells rescued cell survival upon

combined treatment with TRAIL and TSA. Cell confluency was measured prior to treatment

(t=0) or at different time points after treatment over 5 days (t=2-5). The experiment was

performed thrice; the first experiment is shown in Fig. 5D.

F. Immunodetection of PARP1 as well as full-length or cleaved caspase-3 in GH cells depleted

of TNFRSF10B after treatment with TRAIL (16 hrs) or TSA (18 hrs) alone, or both. Combined

treatment resulted in nearly complete cleavage of PARP1 and caspase-3, which was partially

rescued by knocking down TNFRSF10B expression.

G. Cell viability assay in testicular cancer cells treated for 24h with TRAIL or TSA alone, or a

combination of both, using ATP luminometry. For combined treatment the combination index

(CI) was calculated using the Chou and Talalay method. Experiments were performed thrice.

In cases where combined treatment resulted in an inhibitory effect less than 25% of control

levels, CI scores were not determined (NaN) (Miller et al., Sci Signal 2013; 6(294): ra85).

TNFRSF10B expression sensitizes testicular cancer cells towards TRAIL.

Figure S5, continued

G

Vvi

able

cel

ls [

%]

GH cells 1618-K cells

TSA

60

30

0

90

DMSO 50nM 100nM

60

30

0

90

TSA

DMSO 50nM 100nM

control

25ng/ml TRAIL

50ng/ml TRAIL

Combination Index (CI)

0.62

0.660.61

0.52

0.57

0.58

NaN

NaN