1. Supplementary Table 1
Sequences of the primer used for q-PCR analysis
hGAPDH forward, 5’-AGCAATGCCTCCTGCACCACCAAC-3’
hGAPDH reverse, 5’-CCGGAGGGGCCATCCACAGTCT-3’
hPARP1 forward, 5’-TCTACCCCCTGGAGATTGACT-3’
hPARP1 reverse, 5’-GGTCGATCTCATACTCCACCAT-3’
hPARP3 forward, 5’-AACTGGGTAATCGGAAGCTG-3'
hPARP3 reverse, 5'-ACCAGAATGTGGCATGATGC-3'
hPARG forward, 5'-AGCAAGAGACGCTGACATTG-3'
hPARG reverse, 5'-TTCCAGTGCGACTCTCTCCT-3'

2. A
Low risk
High risk
2log PARP1
p 1.0e-06
n=318
n=83
6.0
8.5
8.0
7.5
7.0
6.5
MNSC B C
1.0
0.8
0.6
0.4
0.2
0.0 48 96
144
192
Overall survival
Follow up in months
High PARP1 (n=101)
LowPARP1 (n=300)
p 9.4e-07
MNSC
1.0
0.8
0.6
0.4
0.2
0.0 48 96
144
192
Follow up in months
Event-free survival
High PARP1 (n=101)
LowPARP1 (n=300)
MNSC
p 7.8e-06 D E
1.0
0.8
0.6
0.4
0.2
0.0
Overall survival 48 96
144
192
Follow up in months
p 1.7e-03
High PARP1 (n=82)
LowPARP1 (n=240)
Low Risk
1.0
0.8
0.6
0.4
0.2
0.0
Event-free survival 48 96
144
192
Follow up in months
p 1.1e-03
High PARP1 (n=82)
LowPARP1 (n=240)
Low Risk F G
1.0
0.8
0.6
0.4
0.2
0.0
Overall survival 48 96
144
192
Follow up in months
p 7.0e-04
High PARP1 (n=20)
LowPARP1 (n=63)
MNSC/High Risk
1.0
0.8
0.6
0.4
0.2
0.0 48 96
144
192
Follow up in months
p 1.6e-03
High PARP1 (n=20)
LowPARP1 (n=63)
MNSC/High Risk
Event-free survival
Figure S1
PARP1 expression predicts poor outcome in neuroblastoma patients.
A, Box plot of PARP1 expression relative to risk. B, C, D, E, F, G, Kaplan-Meier curves reporting patients overall and event-free survival probability with respect to PARP1 expression in the MNSC (B, C), low risk (D, E) and MNSC/high risk (F, G) patients subgroups. P value as indicated.

3. A B
2log PARP2
3.5
6.0
5.5
5.0
4.5
4.0
Stage 1
Stage 2
Stage 3
Stage 4
Stage 4S
n=53
n=183
n=121
n=78
n=63
MNSC
MNA
p 1.5e-04
n=401
n=92
2log PARP2
3.5
6.0
5.5
5.0
4.5
4.0 C D
Low Risk
High Risk
p 5.1e-12
n=322
n=176
2log PARP2
3.5
6.0
5.5
5.0
4.5
4.0
Low Risk
High Risk
p 3.3e-08
n=318
n=83
2log PARP2
3.5
6.0
5.5
5.0
4.5
4.0
MNSC
6.5 E F
1.0
0.8
0.6
0.4
0.2
0.0
Overall survival
High PARP2 (n=126)
LowPARP2 (n=372) 48 96
144
192
Follow up in months
p 1.0e-03
1.0
0.8
0.6
0.4
0.2
0.0
Event-free survival
High PARP2 (n=126)
LowPARP2 (n=372) 48 96
144
192
Follow up in months
p 5.9e-03
Figure S2
PARP2 expression, like PARP1, predicts poor prognosis and is associated with stage, MYCN status and risk in NB patients.
A, B, C, D, Box plot of PARP2 expression relative to tumor stage (A), MYCN status (B), and risk classification in the total set (C) or in the MNSC subgroup (D). P values: 5.2e-03 (stage 2 versus stage 4) and 0.02 (stage 4 versus stage 4S). E, F, Overall (E) and event-free (F) survival relative to PARP2 expression in NB patients. P value as indicated.

4. mRNA expression (fold change)B C
MNSC
MNA
Relative PARP2 mRNA expression
0.00
0.02
0.04
0.06
0.08
0.0
0.1
0.2
mRNA expression
Relative
b-Actin
PARP1
MYCN
SK-N-SH
SK-N-MYC *
Genes
MNSC
MNA
p value
PARP1
7,38
4.90E-13
PARP2
4,85
1.50E-04
PARP3
0.06
PARP4
0.15
PARP5a
6,95
2.50E-03
PARP5b
6,14
6.00E-09
PARP6
7,09
4.90E-05
PARP7 3
6.50E-03
PARP8
4,75
8.30E-12
PARP9
3,11
1.10E-03
PARP10
4,41
3.10E-11
PARP11
0.27
PARP12
4,08
1.80E-15
PARP13
1.30E-08
PARP14
2.10E-10
PARP15
9.10E-09
PARP16
4,14
7.60E-48
PARG
4,53
2.80E-08
ARH3
4,43
2.50E-47
TARG1
6,91
5.20E-24 D *
***
PARG
PARP3
mRNA expression (fold change)
2.5
2.0
1.0
0.5
1.5
MYCN+
MYCN-
Color key:
Low
High expression
Figure S3
PARP isoforms expression in neuroblastoma.
A, Heat map diagram of PARP isoforms and PAR degrading enzymes (PARG, TARG1 and ARH3) expression in primary neuroblastoma samples (GSE62564 R2-dataset). Samples are clustered by MYCN status (MNSC, MYCN single copy and MNA, MYCN amplified). Expression levels are indicated by color intensity as shown in the color key legend. P values are reported. B, Real Time q-PCR of PARP2 expression in the MNSC and MNA cell lines reported in Fig. 1. C, Real Time q-PCR and WB analysis of PARP1 expression in the stably transfected SK-N-MYC compared with the control. For qPCRs, the levels of PARP1 and PARP2 mRNA were normalized on GAPDH expression. Data are reported as averages (+/- SD) of two independent experiments (*P<0.05). D, Real time q-PCR of PARG and PARP3 expression in the MYCN+/- SHEP Tet21/N system confirms R2-based expectations. The levels of mRNA were normalized on GAPDH expression. Data are reported as averages (+/- SD) of three (for PARG) and two (for PARP3) independent experiments (* P<0.05; ***P<0.005).

5. A B
PAR
Hsp70
Ola + -
SK-N-SH
Kelly
MYCN-
MYCN+
1.0
1.2
0.6
0.8
0.4
0.2
Colony number (%)
Kelly
SK-N-SH 40 20 80 60
100
120
CTR
5 mM
10 mM ** C D
Colony number (%)
CTR
5 mM
10 mM
MYCN-
MYCN+ **
Time (hours)
Cell number (*10^4)
MYCN- CTR
MYCN+ CTR
MYCN- Ola
MYCN+ Ola 48 72 96
Figure S4
MNA and MYCN-OE cells are more sensitive to PARP inhibitors.
A, WB analysis of PARylation in olaparib treated MNSC, MNA and MYCN-overexpressing cells. Blots were probed with anti-PAR antibody and Hsp70 was used as loading control. B, C Colony formation assay performed on SK-N-SH, Kelly (B) and MYCN+/- (C) neuroblastoma cells after exposure to olaparib or DMSO (CTR) for 24 hours. Data are reported as averages (+/- SD; **P<0.01). D, Viable cell counts performed on MYCN+/- cells treated with or without olaparib for the indicated times. Data are reported as averages (+/- SD) of three independent experiments.

6. A B C D E 7-AAD Figure S5 Olaparib effects on neuroblastoma cells.
Kelly CTR
SK-N-SH CTR
Kelly Ola
SK-N-SH Ola
7-AAD
EdU
# cells C D
Ola - +
SK-N-SH
Kelly
Tail moment (fold ind.)
24 h
15 h
*** +
Kelly
Ola
p53S15P
p53
Chk1S345P
Chk1
Cdc25a
b-actin -
SK-N-SH 3h 6h 9h
12h
24h E
EdU + cells (%) 9 12 24
MYCN+/CTR
MYCN-/CTR
MYCN+/Ola
MYCN-/Ola
Figure S5
Olaparib effects on neuroblastoma cells.
A, Flow cytometry analysis of DNA synthesis (EdU) and DNA content (7-AAD) in indicated cells treated with or without olaparib for 48 hours. Cell cycle profiles are reported in lower panels. The arrowhead indicates the EdU-negative nuclei featuring <4N DNA content. B, Representative images of nuclear morphology in Hoechst stained MYCN+/- cells treated with or without olaparib for 48 hours. (Scale bar: 10μm). C, Neutral comet assay performed in the indicated cells treated with or without olaparib for 15 and 24 hours. Data are reported as averages (+/- SE) of three independent experiments (*** P<0.001). D, WB analysis of whole cell lysates prepared from the indicated cells. Blots were probed with the indicated antibodies. E, Percentage of EdU positive cells in MYCN+/- treated with or without olaparib at indicated times.

7. A B C 1 10
Ola [mM]
Cell cycle distribution (%)
Ola
[mM]
0.01
0.1 1 10
PAR
b-actin
CHKSer345P
CHK1
Ola
[mM] 1 10
b-actin D
0.01
0.1 1 10
0.5 2
PAR
b-actin
Vel [mM]
Tal [mM]
Figure S6
PARP enzymatic inhibition is not sufficient to induce cell cycle dysregulation.
A, WB analysis of PARylation in MYCN-overexpressing cells after 9 hours of olaparib treatment at the indicated concentrations. Blots were probed with anti-PAR antibody and b-actin was used as loading control. MYCN-overexpressing cells treated for 9 hours with or without olaparib at the indicated concentrations were analyzed by flow cytometry for DNA content (B) and by WB for CHK1 phosphorylation (C). D, WB analysis of PARylation in MYCN-overexpressing cells after 48 hours of veliparib (Vel) and talazoparib (Tal) treatment at indicated concentrations. Blots were probed with anti-PAR antibody and anti b-actin as loading control.