Supplementary Data

Methods Oligonucleotide microarray analysis

For microarray analysis, total RNA was isolated from snap-frozen neuroblastoma samples,

and RNA integrity was assessed using the 2100 Bioanalyzer (1). Gene expression profiles

were generated using customized 11K oligonucleotide microarrays (Agilent Technologies) as

dye-flipped dual-color replicates as described elsewhere (1). The array comprised specific

probes for the HDAC family members 1, 2, 3, 6, 7A, 9, and 11. After washing and scanning,

raw microarray data were processed using Agilent’s Feature Extraction Software (Version

7.5.1.) and data normalization was performed using the Linear and LOWESS normalization

algorithm with default parameters. Feature-extracted, normalized data were then imported

into the Rosetta Resolver Gene Expression Analysis System (V5.1; Rosetta Inpharmatics

LLC, Seattle, USA) and data from dye-flipped chip pairs were averaged.

Real time RT-PCR analysis of mRNA

Tumor samples: RT-PCR was performed using the SYBR Green I reagent on the ABI PRISM

7700 Sequence Detection System (Applied Biosystems, Weiterstadt, Germany) as described

elsewhere (2). 5µg total RNA was converted into first-strand cDNA in a volume of 52.5µL.

Target sequences were amplified using standard conditions in a volume of 30µL containing

0.4µL of 1:10 diluted first strand cDNA, 26.8µL of SYBR Green PCR Master Mix (Applied

Biosystems), and 1.4µL of 2.5µmol/L forward and reverse primer each (Thermo Electron).

Primer sequences for HDACs are listed in table S1. Normalization of relative expression

values was performed using the geometric mean of mRNA levels of the control genes SDHA

and HPRT1, which are consistently expressed in primary neuroblastoma (2), and calibrated to

the minimal expression value within the total set of tumors.

Cell lines: Total RNA was isolated from neuroblastoma cell lines with the RNeasy Mini Kit

(Qiagen) according to the manufacturer's instructions. Aliquots of 1µg of total RNA, random

primers and Moloney murine leukaemia virus reverse transcriptase (Invitrogen) were used for

cDNA synthesis. Quantitative PCR was performed with an Applied Biosystems Prism 7000

instrument using SYBR® green master mix from Eurogentec and specific primer pairs for

HDAC2 and 8, GAP43 (growth associated protein 43), MAP2 (microtubule associated protein

2), NTRK1 (neurotrophic tyrosine kinase, receptor, type 1), NTS (neurotensin), NEF-M/L

(neurofilament, medium/light polypeptide), NES (nestin), p21WAF1/CIP1, TUBB3 (tubulin, beta

3), SDHA (succinate dehydrogenase complex, subunit A) and HPRT (hypoxanthine

phosphoribosyltransferase 1). Primer sequences are listed in table S1. Data were analysed

using Applied Biosystems Prism software and the ∆∆CT method. Target gene expression was

normalised against SDHA and HPRT as described above. The difference between mean

threshold PCR cycle values for target and control genes gave the ∆CT value. All reactions

were performed in duplicates and experiments were repeated at least three times.

Cell lines and reagents

Human neuroblastoma cell lines BE(2)-C, SH-SY5Y, Kelly, were purchased from DSMZ or

ECACC, respectively. Human neuroblastoma cells, NGP, SH-EP and WAC2 cells were

generously provided by the laboratory of M. Schwab (3-5), SK-N-BE(2) was obtained from

the laboratory of A. Eggert (6). BE(2)-C, SK-N-BE(2) and SH-SY5Y cells were cultured in

DMEM with L-glutamine and 4.5g/l glucose containing 10% fetal bovine serum and 1%

NEAA. NGP, SH-EP and Kelly were cultured in RPMI 1640 containing 10% fetal bovine

serum. WAC2 were cultured in RPMI 1640 containing 10% fetal bovine serum and 200mg/l

G418.

Western blot analysis

1x105 cells were seeded on 10cm-dishes and 96h after transient transfection with 25nM

siRNA, cells were incubated in lysis buffer (62.5mM Tris/HCl pH6.8, 10% glycerol, 2%

SDS, 1mM DTT). In the case of HDAC inhibitor treatment, 6x105 cells were seeded into

10cm-dishes and lysed 6h after addition of compounds. Protein concentration was determined

by BCATM Protein Assay (Pierce) according to the manufacturer’s protocol. Protein samples

were subjected to SDS-PAGE and transferred to nitrocellulose membrane. The membrane

was blocked in 3% (w/v) non-fat milk in TBS 1h at room temperature and incubated with

primary antibody overnight, followed by incubation with matching horseradish peroxidase-

conjugated secondary antibody at room temperature. Signals were developed with enhanced

chemiluminescence reagents (Amersham) and exposure to X-ray films. The following

antibodies were used: anti-HDAC2 (monoclonal, Epigentek) diluted 1:100, anti-HDAC8

(monoclonal, Epigentek) diluted 1:100, anti-acetyl-Histone H4 (polyclonal, Upstate) diluted

1:2000, anti-acetyl-tubulin (Clone 6-11B-1, Sigma) diluted 1:5000, anti-p21waf1/cip1

(monoclonal, Upstate) diluted 1:1000, anti-actin (Clone AC-15, Sigma) diluted 1:10000, anti-

N-Myc (monoclonal, BD PharMingen) diluted 1:5000 and anti-Trk (C-14, sc-11, Santa Cruz

Biotechnology) diluted 1:200. Actin expression served as loading control. Protein extracts

from primary neuroblastoma tumors from patients with the following characteristics were

used: NB#1: age 63 days, stage 1, MYCN single copy, 1p normal, 11q normal, Shimada

favorable. NB#2: age 245 days, stage 1, MYCN single copy, 1p normal, 11q normal, Shimada

favorable. NB#3: age 5 years, stage 4, MYCN single copy, 1p normal, 11q deletion, Shimada

unfavorable. NB#4: age 5 years, stage 4, MYCN single copy, 1p imbalance, 11q imbalance,

Shimada unfavorable.

Caspase-3-like protease activity assay

Caspase-3-like protease activity was measured with the Caspase-3 Fluorometric Assay

(BioVision) according to the manufacturer’s protocol. 2x105 cells were seeded on 10cm-

dishes and 48h after transfection with 25nM siRNA, cells were collected by trypsinization,

incubated in cell lysis buffer for 10min on ice and then resuspended in reaction buffer (with

10mM DTT) containing AFC-labeled Caspase-3-specific peptide DEVD (50µM). The assay

was performed in black 96-well plates at 37°C. Caspase-3-like activity was determined

fluorometrically using a fluorescence plate reader with a 380nm excitation filter and a 520nm

emission filter. Blank (substrate in buffer without cell extract) values were subtracted and

slope/min values were used to calculate relative Caspase-3-like activity. All experiments were

performed in duplicate and experiments were repeated three times.

HDAC activity assay

Overall HDAC activity was measured essentially as described previously using a cell-based

version of a fluorigenic HDAC assay (7). To measure the decrease in global endogenous

HDAC activity of neuroblastoma cells after siRNA-mediated knockdown, cells were

harvested 48h after transfection and 3x104 cells/well were plated in a 96-well plate. A 2h

cultivation period was followed by a 2.5h incubation period with the cell-permeable

acetylated fluorogenic substrate Boc-Lys(Ac)-AMC (Bachem) in culture medium without

phenol red. Cells were lysed in 50mM Tris/HCl, pH8.0, 137mM NaCl, 2.7mM KCl, 1mM

MgCl2 with 1% (v/v) NP40 and 10mg/ml trypsin. Release of fluorescent AMC which

correlates with deacetylating HDAC enzyme activity was measured in a fluorescence plate

reader with excitation at 380nm and emission at 460nm. Experiments were performed in

triplicate and experiments were repeated three times.

To assess the effect of HDAC inhibitors on endogenous HDACs of neuroblastoma cells, the

HDAC assay described above was employed using 2x104 cells/well cultured for 24h in a 96

well plate. After incubating the cells with the fluorogenic substrate (final concentration

135µM Boc-Lys(Ac)-AMC, Bachem) together with the indicated inhibitors for 3h, cells were

lysed and HDAC activity was determined as described above. Experiments were performed in

triplicate and experiments were repeated three times.

Table S1

Primer Sequences

Gene name Forward Reverse CDKN1A

(p21WAF1/CIP1)

5’-TGGAGACTCTCAGGGTCGAAA-3’ 5’-GGCGTTTGGAGTGGTAGAAATC-3’

GAP43 (8) 5’-ACGACCAAAAGATTGAACAAGATG-3’ 5’-TCCACGGAAGCTAGCCTGAA-3’

HDAC1 5’-TGACGAGTCCTATGAGGCCATT-3’ 5’-CCGCACTAGGCTGGAACATC-3’

HDAC2 5’-TGTGAGATTCCCAATGAGTTGC-3’ 5’-GGTAACATGCGCAAATTTTCAA-3’

HDAC3 5’-CCTCACTGACCGGGTCATG-3’ 5’-ACCTGTGCCAGGGAAGAAGTAA-3’

HDAC4 5’-GAGGTTGAGCGTGAGCAAGAT-3’ 5’-TAGCGGTGGAGGGACATGTAC-3’

HDAC5 5’-GTCTCGGCTCTGCTCAGTGTAGA-3’ 5’-GGCCACTGCGTTGATGTTG-3’

HDAC8 5’-CCAAGAGGGCGATGATGATC-3’ 5’-GTGGCTGGGCAGTCATAACC-3’

HDAC10 5’-ATCTCTTTGAGGATGACCCCAG-3’ 5’-ACTGCGTCTGCATCTGACTCTC-3’

HDAC11 (9) 5’-CAATGGGCATGAGCGAGAC-3' 5’-TGTGGCGGTTGTAGACATCC-3’

HPRT (2) 5’-TGACACTGGCAAAACAATGCA-3’ 5’-GGTCCTTTTCACCAGCAAGCT-3’

MAP2 QT00057358 (Qiagen)

MYCN 5’-CCACGTCCGCTCAAGAGT-3’ 5’-CCCTGAGCGTGAGAAAGCTG-3’

NEF-L (8) 5’-GTGACCAAGCCCGACCTTT-3’ 5’-ATTCCTCAGCGTTCTGCATGT-3’

NEF-M QT0073885 (Qiagen)

NES QT00235781 (Qiagen)

NTRK1 (8) 5’-CAGCCGGCACCGTCTCT-3’ 5’-TCCAGGAACTCAGTGAAGATGAAG-3’

NTS 5’ GCT AGA GAG AGC CCC CTT CAG 3’ 5’ TCA TTC CTG CCA TCA TCT TTC A 3’

SDHA (2) 5’-TGGGAACAAGAGGGCATCTG-3’ 5’-CCACCACTGCATCAAATTCATG-3’

TUBB3 (8) 5’-AGCAAGAACAGCAGCTACTTCGT-3’ 5’-GATGAAGGTGGAGGACATCTTGA-3’

Table S2

Prognostic value of HDAC 1-11 HDAC INSS stage 4 vs. 1 INSS stage 4 vs. 4S

1 P>0,01 P>0,01 2 P>0,01 P>0,01 3 P>0,01 P>0,01 4 P>0,01 P>0,01

5 P>0,01 P>0,01 6 P>0,01 P>0,01 7 P>0,01 P>0,01 8 P<0,001 (***) P<0,01 (**) 9 P>0,01 P>0,01 10 P>0,01 P>0,01 11 P>0,01 P>0,01

Figure Legends

Supplementary Fig. S1

HDAC8 protein expression.

A Immunohistochemical staining of HDAC8 protein in two primary neuroblastoma samples.

Left panel: clinical low risk neuroblastoma (age 6 months, stage 1, MYCN single copy, 1p

normal, 11q normal, Shimada favorable) at low (a) and high (b) magnification. Right panel:

clinical high risk neuroblastoma (age 5 years, stage 4, MYCN single copy, 1p normal, 11q

del, Shimada unfavorable) at low (c) and high (d) magnification. Note the predominant

cytoplasmatic staining of HDAC8 protein in neuroblastoma cells.

B Western Blot showing HDAC8 protein expression in neuroblastoma cell lines (BE(2)-C,

SK-N-BE(2), Kelly, SH-EP) as well as tumor samples (NB#1-4).

Supplementary Fig. S2

Knockdown of HDAC8

A Transfection of 5 siRNAs (siRNA1-5) targeting HDAC8 mRNA in different regions, results

in efficient knockdown of HDAC8 mRNA. SiRNA1 directed against HDAC2 serves as

specificity control, NC-2 = negative control siRNA2. B Transfection with siRNAs targeting

HDAC8 results in strong reduction of HDAC8 protein (44 kDa). NC-1, 2, 3 = negative

control siRNAs.

Supplementary Fig. S3

HDAC2 but not HDAC8 knockdown induces apoptosis in BE(2)-C neuroblastoma cells.

A Upon transfection with siRNA1 targeted against HDAC2 Caspase-3-like activity increases

6-fold compared with transfection of negative control siRNA 2 (NC-2, P=0.0002). In contrast,

knockdown of HDAC8 (siRNA1) did not change Caspase-3 activity. B The amount of

apoptotic nuclei increases 13-fold compared to NC-2 transfection (P<0.0001) following

HDAC2 knockdown. C The number of apoptotic cells in the sub-G1 area of the cell cycle

profile of cells increases significantly to 25% (P=0.0010), as measured by propidium iodide

staining. Untr = untreated control cells, mock = cell treated with transfection reagent only,

NC-2 = cell transfected with negative control siRNA2.

Supplementary Fig. S4

Knockdown of HDAC8 upregulates differentiation markers in NB cell lines.

Expression of MAP2, NEF, and NTRK1 mRNA levels were determined by realtime RT-PCR

following knockdown of HDAC8 (siRNA #1) in Kelly (black bars), SH-EP (open bars) and

SK-N-BE(2) (hatched bars) neuroblastoma cells. mRNA levels were calculated relative to

negative control siRNA (NC-2) transfected cells after normalization to house keeping genes.

Supplementary Fig. S5

Overexpression of HDAC8 results in suppression of neural differentiation.

A HDAC8 overexpression suppresses retinoic acid induced morphological differentiation.

BE(2)-C neuroblastoma cells were stably transfected either with empty vector plasmid or

HDAC8 cDNA containing vectors, respectively. Individual clones were subjected to 10µM

retinoic acid (ATRA) treatment for 7 days. Microscopic photographs of two representative

clones carrying either empty vector controls (a, b) or HDAC8 cDNA expression vectors (c, d)

are shown.

B Quantification of neurite outgrowth of experiments shown in A. Number of neurite-like

extensions per cell exhibiting neurite length of greater then two-fold the cell diameter were

microscopically counted. Mean values of three empty vector clones and three HDAC8

overexpressing clones are shown.

C HDAC8 overexpression suppresses expression of differentiation markers. Basal expression

levels of neurofilament, NTRK1 and p21 mRNA in three empty vector clones and three

HDAC8 overexpressing clones were analyzed using realtime RT-PCR. mRNA expression

levels were calculated relative to negative control transfected cells (NC-2) after normalization

to housekeeping genes. Open bars: empty vector controls, black bars: HDAC8

overexpressing clones.

Supplementary Fig. S6

HDAC8 inhibition has no significant effect on overall HDAC activity in BE(2)-C

neuroblastoma cells.

A HDAC8 selective inhibitor Compound 2 (10 and 20µM) is neither increasing histone 4

(pan-Ac-H4) nor tubulin acetylation (Ac-tubulin) in BE(2)-C cells. Pan-HDAC-inhibitors

(2mM VPA, 0.1µM TSA) increase histone 4 acetylation levels and TSA additionally

increases tubulin acetylation. PBS and DMSO (D) = solvent treated control cells. B

Knockdown of HDAC8 with four different siRNAs (siRNA1, 2, 4, 5) does not increase global

histone 4 acetylation levels in BE(2)-C cells. Knockdown of HDAC2 with four different

siRNAs (siRNA1, 2, 3, 4) increases the acetylation levels of histone 4. In some experiments,

histone H4 acetylation levels appeared lower compared to controls after HDAC8 knockdown.

To rule out compensatory upregulation of other HDAC family members, we investigated the

expression of other class I HDACs 1-3 following knockdown of HDAC8 and found no

changes in mRNA expression levels (data not shown).

Untr = untreated control cells, mock = cells treated with transfection reagent only, NC-1, 2, 3

= cells transfected with negative control siRNAs 1-3. C Knockdown of HDAC2 with siRNA1

significantly decreases overall HDAC activity (p=0.0003), whereas HDAC8 knockdown with

siRNA1 has only marginal effects, which were statistically not significant. HDAC activity is

expressed relative to untreated cells. D The HDAC8 selective inhibitor Compound 2 has no

influence on overall HDAC activity, whereas pan-HDAC-inhibitor (VPA) decreases HDAC

activity. HDAC activity is expressed relative to untreated cells.

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