Lamers et al. BMC Cancer 2012, 12:285 http://www.biomedcentral.com/1471-2407/12/285 added to the cells following manufacturer’s protocol in a concentration of 20 μM. Western blotting Attached and floating cells were harvested on ice 72 h after transduction with shRNA. Cells were lyzed with Laemmlibuffer (20% glycerol, 4% SDS, 100 mM Tris HCl pH 6.8 in mQ). Protein concentrations were quanti- fied with RC-DC protein assay (Bio-Rad, Hercules, USA). Lysates were separated on a 10% SDS-Page gel and transferred onto a blotting membrane (Millipore, IPFL00010). Blocking and incubation were performed in OBB according to manufacturer’s protocol (LI-COR). Primary antibodies used were anti-BIRC6 (Abcam, ab19609), anti-DIABLO (Abcam, ab32023), anti-PARP (Cell Signaling: 9542) and anti-BCL2 (Cell Signalling; 2872). Protein loading was checked by anti-β-actin (Abcam, ab6276) or anti-α-tubulin (Sigma, T5168). The secondary antibodies used were provided by LI-COR. Proteins were visualized with the Odyssey bioanalyzer (LI-COR). In cell western Cells were fixed with 4% paraformaldehyde for 20 min 48 h after transduction with BIRC6 shRNA. Blocking and incubation were performed in OBB according to manufacturer’s protocol (LI-COR). Primary antibodies used were anti-BIRC6 (BD Biosciences, 611193) and anti-β-actin mouse monoclonal (Abcam, ab6276). Pro- teins were visualized with the Odyssey bioanalyzer (LI- COR) and quantified and corrected for β-actin using the Odyssey software. Immunofluorescence Cells were grown on glass slides in 6-well plates and were fixed with 4% paraformaldehyde in PBS 48 h after trans- duction. We used anti-DIABLO (Abcam, ab32023) as a primary antibody, and anti-rabbit (Alexa, 11012) as a sec- ondary antibody. Mitochondria were stained using Mito- tracker (Invitrogen, M22426). Antibodies were dissolved in 5% ELK in PBS/0.2% tween-20. Slides were stained with DAPI (1:1000) in vectashield (Vector Laboratories). Cell fractionation Protein was harvested and fractionated using the Subcel- lular Proteome Extraction Kit according to manufac- turer’s protocol (Novagen, 539790). Fraction I (cytosol) and II (membrane/organelle) were used for Western blot. Co-immunoprecipitation Cells were lyzed in a buffer containing 150 mM NaCl, 50 mM Hepes, 5 mM EDTA, 0.3% NP-40, 10 mM β-glycerophosphate, 6% glycerol, protease inhibitors (Complete mini, Roche) and Phosphatase inhibitors Page 4 of 10 (5 mM NaF, 1 mM Na2VO3). The antibody used for IP was anti-BIRC6 (Abcam, ab19609); negative controls were anti-flag (Cell Signaling, 2368) and protein with- out antibody. Protein-G agarose beads (Roche) and antibody were incubated for pre-coupling overnight after which lysates were added and incubated over- night. Immunocomplexes were washed, heated at 95°C for 10 min and loaded on a gel for Western blot. Primary antibodies used were anti-BIRC6 and anti- DIABLO (Abcam; ab32023). Blots were incubated overnight with primary antibodies, after which a one hour incubation step with anti-rabbit IgG (Cell Sig- naling; 3678) was performed followed by incubation with the secondary antibody provided by LI-COR. MTT-assay Cells were seeded at 30% confluence in a 48-well plate, transduced with BIRC6 shRNA and after 24 h treated with ZVDVAD. Seventy-two hours after treatment, 25 μl of Thiazolyl blue tetrazolium bromide (MTT, Sigma M2128) was added. After 4–6 h of incubation, 250 μl of 10% SDS, 0.01 M HCl was added to stop the reaction. The absorbance was measured at 570 nm and 720 nm using a plate-reader (Biotek). Results Gain of BIRC5 and BIRC6 and loss of CASP9 in neuroblastoma tumors To identify patterns in the aberrations of genes involved in intrinsic apoptotic signaling, we combined high throughput analysis of DNA copy number and mRNA expression of these genes. We included all 31 genes that are directly involved in the mitochondrial apoptotic path- way and their downstream target genes (Table 1). Array CGH data of 87 primary neuroblastoma tumor samples were analyzed for copy number variations of the 31 genes included in our intrinsic apoptotic gene panel. Binary segmentation data was used to score the DNA copy number variations and they were subsequently confirmed using log fold data from SNP array analyses of the same tumors. Only three genes show DNA copy number aber- rations at a frequency above 10% (Figure 1a). The BIRC5 gene, which is located in the smallest region of overlap (SRO) of gain of chromosomal band 17q25 [12], is gained in 49% of the tumors. CASP9 is located at the SRO of deletions of 1p36 [12] and it is lost in 30% of the tumors. BIRC6, which is located on 2p22, is gained in 24% of the neuroblastoma tumors. Distal chromosome 2p is a known region of gain in neuroblastoma. BIRC6 is not located in the SRO of this gained region, but is gained in 84% of the tumors with 2p gain (Figure 1b). Of these three genes, BIRC6 was not studied before in neuroblast- oma. We therefore investigated whether the gain of BIRC6 resulted in aberrant expression. We compared