Lamers et al. BMC Cancer 2012, 12:285 http://www.biomedcentral.com/1471-2407/12/285 variations with the Infinium II assay on Human370/660- quad arrays containing > 370 000/> 660 000 markers and run on the Illumina Beadstation (Swegene Centre for Integrative Biology, Lund University – SCIBLU, Swe- den) according to the manufacturer’s recommendations. Raw data were processed using Illumina’s BeadStudio software suite (Genotyping module 3.0), producing re- port files containing normalized intensity data and SNP genotypes. Subsequently, log 2 Ratio and B-allele fre- quency data were imported into the R2 web application for detailed analysis and comparison with the CGH and expression data. Bioinformatics All data were analyzed using the R2 web application, which is publicly available at http://r2.amc.nl. The expres- sion data were normalized with the MAS5.0 algorithm within the GCOS program of Affymetrix Inc. Target in- tensity was set to 100. For scoring genomic aberrations of the 31 included genes (see below and Table 1), we consid- ered CGH aberrant if the logfold value was more than 0.45 for gain or less than −0.45 for loss and if a breaking point was clearly visible. We excluded whole chromosome gains or losses. Also, the detected gains or losses had to be confirmed by SNP array. Cell lines All cell lines were grown in Dulbecco Modified Eagle Medium (DMEM), supplemented with 10% fetal calf serum, 10 mM L-glutamine, 10 U/ml penicillin/strepto- mycin, Non Essential Amino Acids (1x) and 10 μg/ml streptomycin. Cells were maintained at 37°C under 5% CO2. For primary references of these cell lines, see Molenaar et al. [35]. Lentiviral shRNA production and transduction Lentiviral particles were produced in HEK293T cells by co-transfection of lentiviral vector containing the short hairpin RNA (shRNA) with lentiviral packaging plas- mids pMD2G, pRRE and pRSV/REV using FuGene HD. Supernatant of the HEK293T cells was harvested at 48 and 72 h after transfection, which was purified by filtration and ultracentrifugation. The concentration was determined by a p24 ELISA. Cells were plated at a 10% confluence. After 24 h cells were transduced with lentiviral DIABLO shRNA (Sigma, ‘E8’: TRCN0000004511 and ‘E9’: TRCN0000004512) or BIRC6 shRNA (Sigma, ‘C7’: TRCN0000004157 and ‘C11’: TRCN0000004161) in various concentrations (Multiplicity of infection (MOI): 1–3). SHC-002 shRNA (non-targeting shRNA: CAACAAGATGAAGAGCAC- CAA) was used as a negative control. Medium was refreshed 24 h after transduction and puromycin was added to select for transduced cells. Protein was Table 1 Selected genes in the intrinsic apoptotic pathway Gene family or category Anti-apoptotic members of BCL2 family Page 3 of 10 Gene (alias) BCL2 MCL1 BCL2L1 (BCLXL) BCL2L2 (BCLW) BCL2A1 BCL2L10 BBC3 (PUMA) BCL2L11 (BIM) PMAIP1 (NOXA) BID BAD BIK HRK BCL2L14 BMF BAX BAK1 DIABLO CYCS APAF1 CASP3 CASP6 CASP7 CASP9 NAIP XIAP BIRC5 BIRC6 BIRC7 TP53 CASP2 Pro-apoptotic members of BCL2 family Mitochondrial permeabilization Cytoplasmic pro-apoptotic genes Caspases, activated by mitochondrial apoptotic pathway IAPs Activation of mitochondrial apoptotic pathway harvested 72 h after transduction and analyzed by Western blot. Compounds ABT263, a small molecule BCL2 inhibitor, was dissolved in DMSO to a concentration of 20 mM as stock solu- tion. A final concentration of 200 nM ABT263 was used. For the experiment using this compound we chose SJNB12 instead of IMR32 or SKNSH for its high expres- sion of BCL2 and its subsequent high sensitivity to this compound [33]. Z-Val-Asp(OMe)-Val-Ala-Asp(OMe)-FMK (ZVDVAD- FMK, a widely used CASP2 inhibitor; R&D systems) was