SUPPLEMENTARY METHODS
Neuroblastoma patient samples and statistical analyses
Clinical data for 477 neuroblastoma patients described in a study by Kocak et al. (the Kocak cohort)(5) was matched to publically available microarray data. The microarray data was downloaded from R2 microarray analysis and visualization platform (http://r2.amc.nl)(6). Samples from the Children’s Oncology Group (COG) USA were previously described (7). In brief, a cohort of 197 patients enrolled onto COG Neuroblastoma Biology Study 9047 was obtained from the COG Tumor Bank (“COG” cohort). This study population is representative of patients with neuroblastoma in general with respect to well-established prognostic indicators, including MYCN amplification status, age at diagnosis, and tumor stage (8). The protocol was approved by individual institutional review boards and informed consent was obtained for every patient registered in the study.
Kaplan-Meier curves of event-free and overall survival of the Kocak and COG cohorts were compared using the log-rank test. Univariate and multivariate Cox Proportional-Hazard regression models were used to test whether gene expression levels function as prognostic indicators for overall survival (OS) and event-free survival (EFS) of patients in the Kocak and COG cohorts. Known prognostic indicators, including age (<18 months vs 18 months), tumor stage (stage 1, 2, 4s vs stage 3,4), and MYCN status (amplified vs normal) were fitted into the multivariate model.
Quantitative Real-time PCR analysis of gene expression
Total RNA was isolated from cell lines using the RNeasy Plus Mini Prep kit (Qiagen, Germany) according to the manufacturer’s instructions. cDNA was synthesized of by reverse transcription of 1 µg of RNA using SuperScript®III reverse transcriptase (Invitrogen, CA, USA). qRT-PCR reactions were performed using iQTM SYBR Green Supermix (Bio-Rad, CA, USA) and an ABI7900HT real-time PCR machine (Applied Biosystems, CA, USA). The PCR program was: 90°C for 10 minutes followed by 40 cycles of denaturation at 94°C for 1 second, annealing at 58°C for dyskerin and snoRNAs, 60°C for hTERT or 58oC for hTR for 30 seconds, then extension at 72°C for 1 minute and 4°C hold. Each sample was run in duplicate. Data was analyzed using SDS 2.2 software (Applied Biosystems, USA) with a threshold cycle (Ct) used to calculate relative gene expression. Gene expression was normalized to the house keeping gene ABL; ΔCt = Ct Sample – Ct ABL and expressed relative to gene expression levels in HeLa cells according to the formula; Relative gene expression = 2-ΔΔCt, where ΔΔCt = ΔCt – ΔCt HeLa. The nucleotide sequences of primers for DKC1, hTR, TERT and ABL and are listed in Supplementary Table 2. Controls were HeLa cervical cancer cells, which are known to be telomerase positive, normal myofibroblasts (MRC-5 and WI-38) and normal bone marrow endothelial cells (BMEC).
RNA was prepared from tumor specimens using the RNeasy extraction kit (Qiagen), reverse transcribed with Moloney Murine Leukemia Virus reverse transcriptase (Life Technologies, Melbourne, Australia) as previously described (7) and applied to custom-made Taqman low-density array (TLDA) cards (Applied Biosystems). TLDA cards were loaded with 125 ng cDNA (RNA equivalent) per loading port and cards were run on an ABI 7900HT Sequence Detection System (Applied Biosystems). Relative expression of each gene was calculated by the (delta-delta) Ct method using the RQ Manager Program (Applied Biosystems) as the geometric mean of the expression values against each of the three control genes, control genes HPRT, GUSB and PPIA (9). Primer and probe sequences are listed in Supplementary Table 2.
Western blot analysis
Protein was extracted from whole cell lysates by incubation in TRAPeze-CHAPS lysis buffer (Merck Millipore, USA) enriched with protease inhibitor (Roche, Germany) on ice for 30 minutes. Twenty µg of protein lysate was suspended in NuPAGE Lithium-Dodecyl-Sulfate Sample Buffer with NuPAGE Reducing Buffer (Invitrogen, CA, USA) and denatured at 70°C for 10 minutes before electrophoresis through precast 4-12% bis-tris polyacrylamide Criterion gels (Bio-Rad, USA) and subsequent transfer to methanol-activated Polyvinylidene Fluoride (PVDF) membrane (Millipore, MA, USA). Membranes were blocked by overnight by incubation at 4ºC in 5% non-fat dairy milk/Tris-buffered saline/0.5% Tween-20 (TBST; Sigma-Aldrich), then washed in TBST and incubated with primary antibody for an hour at room temperature (for detection of dyskerin and GAPDH proteins). The antibodies used are listed below. The membranes were washed in TBST prior to incubation with HRP-conjugated donkey anti-rabbit or horse anti-mouse secondary antibodies (GE Healthcare) for 1 hour at room temperature and proteins visualized using West Femto Supersignal (Pierce) and autoradiography on Fuji HR-T Medical X ray Film (Fujifilm, Tokyo, Japan) according to the manufacturer’s instructions. For detection of a second protein, antibodies were stripped from membranes using Restore Western Blot Stripping Buffer (Pierce) according to the manufacturer’s instructions.
Antibodies for Western blot and chromatin immunoprecipitation
Antibodies for western blot: dyskerin rabbit polyclonal antibody, generated against the synthetic dyskerin peptide H-CHGKPTDSTPATWKQEY-NH2 (corresponding to amino acids 401 to 416), GAPDH mouse monoclonal antibody (Abcam, lot #GR57834-1), p21cip1 mouse monoclonal antibody (BD Biosciences, lot #30449) and p53 mouse monoclonal antibody (Santa-Cruz Biotechnology, lot #D2908). Antibodies for chromatin immunoprecipitation were N-Myc (sc53993, Santa Cruz Biotechnology), Max (sc197, Santa Cruz Biotechnology) and c-Myc (sc764, Santa Cruz Biotechnology).
Telomere length analysis
Mean telomere length was measured using 2.0 mg of genomic DNA and the TeloTAGGG Telomere Length Assay kit (Roche Mannheim, Germany) as previously described (10). Briefly, DNA was digested with HinfI and RsaI and then fractionated through a 0.7% agarose gel for 26 hours at 65 volts and transferred to a nylon membrane. The Membrane was hybridized to 1:5,000 dilution of the telomere probe and visualized using CDP-star substrate (Sigma-Aldrich), with subsequent exposure of the membrane to x-ray film.
Detection of ribosomal proteins by immunofluorescence
siRNA transfected NB69 cells cultured on glass coverslips were fixed with 4% paraformaldehyde in PBS for 15 minutes, then washed with PBS and permeabilized with 0.1% Triton X-100/PBS for 10 minutes. After blocking for 50 minutes with 10% FCS / PBS, indirect immunofluorescence staining was performed using anti-rpl11 (Abcam; ab86863) or anti-rpl5 (Abcam; ab79352) diluted in blocking solution for 50 minutes. Cells were washed with PBS and stained with secondary antibody (Life Technologies; A-11012) for 50 minutes. Coverslips were mounted in Prolong-Gold (Life Technologies; P36930) and images visualized and captured using a Zeiss Axiovert 200M inverted microscope with a LCI Plan-Neolfluar 63x/1.3 objective and an AxioCam HRm camera (Carl Zeiss Inc., Oberkochen, Germany)
Cell cycle analysis
Aliquots of 5 × 105 cells were washed in cold phosphate-buffered saline (PBS) and fixed in 70% ethanol at 4°C overnight. The cells were then permeabilized by incubation in 1 ml of 0.5% Triton-X/0.1% bovine serum albumin (BSA)/PBS for 30 minutes at room temperature, then washed, resuspended in 400 µl of 25 µg/ml propidium iodide (Sigma-Aldrich) and 100 µg/ml DNAse-free RNAse (Roche, Germany) in 0.1% BSA/PBS and incubated in the dark for 30 minutes at room temperature. The cell suspension was then washed and resuspended in 400 µl 0.1% BSA/PBS for DNA content analysis using FL-2A channel of a FACSCalibur flow cytometer. Singlet populations were identified using the FL2-A and FL2-W parameters and analyzed using ModFit LT version 3.0 software (Verity Software House) and FlowJo software (BD Biosciences, USA). Dead cells and debris were excluded from the analysis by gating on FSC and SSC parameters.
Viral vector-mediated gene suppression
For stable suppression of dyskerin, miR30-styled shRNA targeting the DKC1 gene was synthesized (Sigma-Aldrich) and cloned into the LMS retroviral vector backbone, which co-expresses the green fluorescence protein (GFP) (11). An LMS vector harboring a non-silencing control oligonucleotide (NS) was used as a control (1). Ectopic hTR was expressed from the MND-hTR retroviral vector (2). The empty vector backbone, MND that expresses the neomycin resistance gene (only) was used as a control. Expression of p53 was silenced using the previously described Babepuro-shRNA vector, with GFPshRNA expressed as a control (3). Replication defective retrovirus was generated by transfection of Phoenix-A cells according to our previously described methods (12). Transduced cells were selected by fluorescence-activated cell sorting (FACS) for GFP expression (LMS-based vectors) or by growth in either 0.5 mg/mL genetecin (MND vectors) or 0.5 mg/mL puromycin (Babepuro-shRNA vectors). Lentiviral vectors encoding doxycycline-inducible shRNA targeting DKC1 or a control shRNA (NS) were constructed from the Fh1t-UTG vector backbone, which constitutively co-expresses GFP (13). Lentiviral vector particles were produced by co-transfection of HEK293T cells with the packaging constructs pKGP, pRT and pVSV-G using Lipofectamine 2000 (Invitrogen). Supernatant containing viral particles were collected 24 hours after transfection. To induce shRNA expression, 1 µg/ml doxycycline-hyclate (dox; Sigma-Aldrich) was added to the growth medium every 3 days. Transduction with lentiviral and retroviral vectors was determined by assessment of GFP expression on a FACSCalibur flow cytometer (BD Biosciences, USA).
Assessment of tumorigenicity
Female 6-8 week old BALB/c nu/nu mice (Animal Resource Centre; WA, Australia) were inoculated by subcutaneous injection of 2.0 × 106 cells suspended in 200 µl DMEM into the hind flank. Once the tumors reached a palpable size, mice were switched from regular diet to food laced with 600 mg/kg doxycycline (Specialty Feeds, WA, Australia) to induce vector expression. Tumors were measured in three dimensions every two days using calipers, and mice were sacrificed before tumor volume reached 1.5 cm3. Tumor volume was calculated as: Volume (mm3) = [Length (mm) × Width (mm) × Height (mm)] / 2.
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