DNA extracted from cancer specimens and normal tissue was labeled and hybridized to the Affymetrix 250K Sty I array to obtain signal intensities and genotype calls. Signal intensities were normalized against data from 1480 normal samples. Copy-number profiles were inferred using GLAD48 (link) and changes of > 0.1 copies in either direction were called SCNAs. The significance of focal SCNAs (covering < 0.5 chromosome arms) was determined using GISTIC18 (link), with modifications to score SCNAs directly proportional to amplitude and to allow summation of non-overlapping deletions affecting the same gene. Peak region boundaries were determined so that the change in the GISTIC score from peak to boundary had < 5% likelihood of occurring by random fluctuation. P-values for Figures 2b and 4 were determined by comparing the gene densities of SCNAs and fraction overlap of peak regions respectively to the same quantities calculated from random permutations of the locations of these SCNAs and peak regions. RNAi was performed by inducible and stable expression of shRNA lentiviral vectors and by siRNA transfection. Proliferation in inducible shRNA experiments was measured in triplicate every half-hour on 96-well plates by a real time electric sensing system (ACEA Bioscience) and in stable shRNA expression and siRNA transfection experiments by CellTiterGlo (Promega). Apoptosis was measured by immunoblot against cleaved PARP and FACS analysis of cells stained with antibody to annexin V and propidium iodide. Tumor growth in nude mice was measured by caliper twice weekly. Expression of MYC, MCL1, and BCL2L1 was performed with retroviral vectors in lung epithelial cells immortalized by introduction of SV40 and hTERT49 (link).
Full methods are described inSupplementary Methods .
Full methods are described in