1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
SUPPORTING INFORMATION
Real time PCR primers:
Mouse Primers:
Gene: GAPDH
Forward: 5’ CAACAGCAACTCCCACTCTTCC 3’
Reserve: 5’ ATGTAGGCCATGAGGTCCACC 3’
Gene: BAMBI
Forward: 5’ GCCACTCCAGCTACTTCTTC 3’
Reserve: 5’ CACATGTAACCTGTTGCCAC 3’
Gene: DKK1
Forward: 5’ GAGGGGAAATTGAGGAAAGC 3’
Reserve: 5’ CCTTCTTGTCCTTTGGTGTG 3’
Gene: DKK2
Forward: 5’ ACTCTTCCAAAGCCAGACTC 3’
Reserve: 5’ GCATTCCAATCCAGGTTTCC 3’
Gene: c-Myc
Forward: 5’ TGATCCTCAAAAAAGCCACC 3’
Reserve: CGA AGCTGTTCGAGTTTGTG 3’
Gene: IGFII
Forward: 5’ ACACACACACACACACACAC 3’
Reserve: 5’ TGGGATGGGCCTTGTTTTGG 3’
Gene: EpCAM
Forward: 5’ AGGGGCGATCCAGAACAACG 3’
Reserve: 5’ ATGGTCGTAGGGGCTTTCTC 3’
Gene: RPA2
Forward: 5’ TCAGTGGGTTGACACGGATG 3’
Reserve: 5’ TGATCTTAAAGGCCACCAAGC 3’
Gene: CCNA1
Forward: 5’ TCGCTACCTTCGAGAAGCTG 3’
Reserve: 5’ ATTCTTCCCCAACCTCCACC 3’
Gene: CCND2
Forward: 5’ AAGAAAGCACTCCCTGACTG 3’
Reserve: 5’ TCCTCAAATGCCATCCCCTC 3’
Gene DLK1
Forward: 5’ GCTTCGCAAGAAGAAGAACC 3’
Reverse: 3’ AGATCTCCTCATCACCAGCC 3’
Woodchuck Primers:
Gene: GAPDH
Forward: 5’ TTTGGCAATGTGGAAGGAC 3’
Reserve: 5’ TGAGGGAAGATATTCTGGGC 3’
Gene: BAMBI
Forward: 5’ GTAGCAGAAACCTCATCACC 3’
Reserve: 5’ AGGCCAACATAATCAGCAAC 3’
Gene: DKK1
Forward: 5’ GTCAGCTCAATCCTAAGGATG 3’
Reserve: 5’ ATTTACTGCAAACACAGGGG 3’
Gene: c-Myc
Forward: 5’ ACAACGAAAAGGCCCCCAAG 3’
Reserve: 5’ TAACTGTTCTCGCCGCTTCC 3’
Gene: EpCAM
Forward: 5’ CCGAAGAACTGACAAGGATAC 3’
Reserve: 5’ GCAGTCTGCAAACTTTGAAC 3’
2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
Gene: IGFII
Forward: 5’ GGGCAAGTTTTTCCAATATGAC 3’
Reserve: 5’ CCTCTCTGAACGCTTCAAG 3’
Gene: MCM4
Forward: 5’ TGTTTTCCAGTCCTCCTCAG 3’
Reserve: 5’ TACCCTAACCCCACTCCTTG 3’
Gene: MCM5
Forward: 5’ TGCTGCCAACTCAGTGTTCG 3’
Reserve: 5’ TCTCCTCGTTGTGCTCATCC 3’
Gene: MCM6
Forward: 5’ GCAGTTCAAATACACACAGCC 3’
Reserve: 5’ TGAGTCTCCTGAATACGAACC 3’
Gene: TYMS
Forward: 5’ AATTTCCTCTGCTGACAACC 3’
Reserve: 5’ GCATCCCAGATTTTCACTCC 3’
Gene: PLK1
Forward: 5’ TGCAGCACTTACACAGTGTC 3’
Reserve: 5’ TTTGTCGGAGTAGTCCACCC 3’
Gene: CCND2
Forward: 5’ TCTTTCCCTCCTTCTCACCAC 3’
Reverse: 5’ AGGACCAACTTAGTCCCCAC 3’
Gene: RPA2
Forward: 5’AGGGATGTAAACCAGGATGG 3’
Reverse: 5’ AGAAACTCCCAGGTGTTAGG 3’
Human Primers:
Gene: GAPDH
Forward: 5’ GACCTGCCGTCTAGAAAAAC 3’
Reserve: 5’ TTGAAGTCAGAGGAGACCAC 3’
Gene: BAMBI
Forward: 5’ GTATCAGCATGATGGTAGCAG 3’
Reserve: 5’ AGCAACACTAAAATCAGCCC 3’
Gene: DKK1
Forward: 5’ TGAGTCCTTCTGAGATGATGG 3’
Reserve: 5’ TGAGAACCGAGTTCAAGGTG 3’
Gene: DKK2
Forward: 5’ CTGAAAGCATCTTAACCCCTC 3’
Reserve: 5’ CATCTTAGTGTGTGGTCTTCC 3’
Gene: c-Myc
Forward: 5’ GCCACGTCTCCACACATCAG 3’
Reserve: 5’ TCTTGGCAGCAGGATAGTCCTT 3’
Gene: EpCAM
Forward: 5’ CAGAAGAACAGACAAGGACAC 3’
Reserve: 5’ TGCAGTCCGCAAACTTTTAC 3’
Gene: IGFII
Forward: 5’ TGGAGACGTACTGTGCTAC 3’
Reserve: 5’ CCAGGTGTCATATTGGAAGAA 3’
Gene: RPA2
Forward: 5’ GCTTGTCCAAGACCTGAAGG 3’
Reserve: 5’ ACAGTAGAATAGATGTGCCCC 3’
Gene: CCNA1
Forward: 5’ AAGAAAGCACTCCCTGACTG 3’
Reserve: 5’ TCCTCAAATGCCATCCCCTC 3’
Gene: CCND2
Forward: 5’ AAGTTTGCCATGTACCCACC 3’
3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
Reserve: 5’ TTAGCCAGCAGCTCAGTCAG 3’
Gene: PLK1
Forward: 5’ AAGTCTCTGCTGCTCAAGCC 3’
Reserve: 5’ ACGAACACGAAGTCGTTGTC 3’
Gene: TYMS
Forward: 5’ GAATTCCCTCTGCTGACAAC 3’
Reserve: 5’ GCATCCCAGATTTTCACTCC 3’
Gene: PCNA
Forward: 5’ TCGATAAAGAGGAGGAAGCTG 3’
Reserve: 5’ CATACTGAGTGTCACCGTTG 3’
Gene: MCM4
Forward: 5’ CCTCTCGTAAACGGAAAGAAG 3’
Reserve: 5’ TGCTATGTCAGATTGTCCCC 3’
Gene: MCM5
Forward: 5’ TGCTGCCAACTCAGTGTTC 3’
Reserve: 5’ TCTCCTCATTHTGCTCATCC 3’
Gene: MCM6
Forward: 5’ ACCAGACACAAGATTCGAGAG 3’
Reserve: 5’ TCCAAGCACAGAAAAGTTCC 3’
Gene: DLK1
Forward: 5’ TGTCCAACCTGCGCTACAAC 3’
Reverse: 5’ TGCTGAAGGTGGTCATGTCG 3’
ChIP Assay Primers
Gene: BAMBI
Forward: 5’ CCCTGCAAGCCCTAATAAATG 3’
Reverse 5’ CAATTAATCACCACCACCACC 3’
Gene: CCND2
Forward: 5’ TCTCCAACATCCACCCCTTC 3’
Reverse: 5’ TCGCCAAACCAGGGATAAAG 3’
Gene: DKK2
Forward: 5’ GTAGGAAAGGTGAGAGGCAG 3’
Reverse: 5’ CCCCAATTCAAGAGTGACAAAG 3’
Gene: EpCAM
Forward: 5’ACCCAAAGCCCTCTTCTGAC 3’
Reverse 5’ TGGCAGCTTACTTTCCAATACC 3’
Gene: IGFII
Forward: 5’ ACCCAACAGCAACAACAAAC 3’
Reverse: 5’ AATGGGGGGCATCTTGATCC 3’
Gene: c-Myc
Forward: 5’ TTCCCCAGCCTTAGAGAGAC 3’
Reverse: 5’ GTTCTTGCCCTGCGTATATC 3’
Gene: DLK1
Forward: 5’ AGGGGTAGTATAGGTAGGAGGATT 3’
Reverse: 5’ AAATCTCAAAAACCAAACCAAAC 3’
Gene: RPA2
Forward: 5’ GTTGCTGGGAATTGAACTCG 3’
Reverse 5’ TTCACACACACCCCATCTCC 3’
50
51
Supporting Table 1:
52
Expression of indicated genes during oncogenic transformation of X-expressing cells. P2-P5 cultures are derived by
4
1
consecutive passages of partially polyploid pX-expressing cells, isolated by live cell-sorting from the immortalized
2
4pX-1 cell line, as described (14). P2 and P3 cultures are not transformed, whereas P4 and P5 cultures are pX-
3
transformed cells (14). 4pX-1-Suz12kd is a Suz12 knockdown cell line (16), used as positive control. Fold induction
4
of indicated genes was quantified relative to expression in untransformed 4pX-1 cells. Results are from at least three
5
independent RNA preparations used in real-time PCR reactions. Each PCR reaction was performed in triplicates and
6
quantified relative to GAPDH used as internal control, +/- standard error of the mean.
7
Supporting Table 2:
8
Fold induction of indicated genes in the liver of 4-month old X monotransgenics, c-myc monotransgenics and X/c-
9
myc bitransgenics in comparison to WT mice of the same age, quantified by real-time PCR. Results are from at least
10
three independent RNA preparations used in real-time PCR reactions. Each PCR reaction was performed in
11
triplicates and quantification was relative to GAPDH used as internal control, +/- standard error of the mean.
12
.
13
Supporting Table 3:
14
Fold induction of the proliferation gene cluster and Suz12-repressed genes quantified in tumor vs peri-tumoral
15
paired tissues from liver of individual WHV-infected animals. The woodchuck identification number is from the
16
study by Jacob et al (39). Results are from at least three independent RNA preparations used in real-time PCR
17
reactions. Each PCR reaction was performed in triplicates and quantification was relative to GAPDH used as
18
internal control, +/- standard error of the mean.
19
20
Supporting Figure 1:
21
Chromatin immunoprecipitation (ChIP) assays with Suz12 antibody or IgG as indicated in 4pX-1 cells, pX-
22
transformed cells (14), and 4pX-1-Suz12kd cells (16). The ChIP-derived DNA was amplified by PCR with primer
23
pairs (see Supporting information) for indicated genes and PCR products were analyzed by agarose gel
24
electrophoresis.
25
26
Supporting Figure 2:
27
HepAD38 cells that support HBV replication following removal of tetracycline (35), were grown with (+) or
28
without (-) tetracycline for 10 days, i.e. without (-) or with (+) HBV replication, respectively. A. 250 nM or 500 nM
5
1
BI 2536 added on day 9 for 24 h (day 1) or day 8 for 48 h (day 2), respectively, as indicated, prior to cell harvesting.
2
WCE isolated without (-) or with (+) addition of BI 2536 were immunoblotted for HBV core antigen, caspase 3 and
3
cleaved/active caspase 3. B. Immunoblots of active Phospho-Plk1 (T210) and Plk1 using WCE isolated from
4
HepAD38 cells grown in the presence of 250 nM BI 2536 for 2 days or 500 nM BI 2536 for 1 day, as indicated. C.
5
250 nM or 500 nM BI 2536 added on day 8 for 48 h (2 days) or day 9 for 24 h (1 day), respectively, as indicated,
6
prior to isolation of total RNA. Expression of viral RNA was quantified by real-time PCR employing primer pairs
7
described by Zhang et al, Hepatology 53, 1476-1485, 2011. Results are the average from two independent WCE
8
preparations. Error bars indicate +/- standard error of the mean. D. Quantification of protein levels of Suz12 and
9
Znf198 from immunoblots shown in Fig. 5B, using lysates of HepAD38 cells grown with (+) or without (-)
10
tetracycline for 10 days and treated for 24 h prior to harvesting with 500 nM BI2536. Quantification of indicated
11
proteins is by ImageJ software vs. actin. Results are the average from two independent WCE preparations. Error bars
12
indicate +/- standard error of the mean.
13
14
Supporting Figure 3. Analysis of microarray data from the study by Boyault et al (29). Relative induction of
15
indicated genes is individual liver tumors with high HBV titer (H), low HBV titer (L), no HBV titer (N), and non-
16
tumor normal liver (n).
17