the Alamar blue score in IGF1R-overexpressing cells to the score for MCF10A cells grown in Matrigel was calculated. This ratio was used to select candidates for testing additional siRNA reagents. Assessment of cell death Cell death in suspension cultures was assessed using the Cell Death ELISA kit (Roche Diagnostics).according to the manufac- turer’s instructions. Soft agar colony assays The bottom layer of soft agar contained 0.6% agar with DMEM and 5% serum. Fifteen thousand cells were seeded per well on the top layer that contained 0.3% agar in MEGM. Cells were seeded in triplicate wells of six-well plates. Wells were re-fed every two weeks and colonies were counted at four weeks post-plating. At each time point, an image of each well was taken at 66 magnification and processed using the software Image J. Colonies larger than 50 sq. pixels were counted. SNP array analysis Breast tumors for SNP array analysis were collected from de- identified cases using protocol #98-229 approved by the Institutional Review Board of the Dana-Farber Cancer Institute. SNP array analyses were performed by the Dana-Farber Microarray Core and by the Broad Institute using Affymetrix 250K StyI arrays, according to methods described by Nikolsky et al [30]. The raw .CEL files were normalized and copy number of each SNP was determined using the GenePattern software [30]. Raw SNP copy numbers were smoothed by a segmentation algorithm using the DNA copy package (available at www.bioconductor.org). Copy numbers of each gene were estimated by averaging copy numbers from all SNPs found within the gene structure and flanking 100-kbp regions. All of the raw data were deposited into Oncomine and Gene Expression Omnibus (GSE19399) and are publicly available: (https://www.oncomine. com/resource/login.html and http://www.ncbi.nlm.nih.gov/sites/ entrez?db = gds&term = GSE19399[Accession]&cmd = search). FISH analysis of tumors A fosmid probe specific for PTK6 and a Chromosome 20 centromere probe were obtained from CHORI/BacPac library. Hybridization and analyses were performed by the Dana-Farber/ Harvard Cancer Center Cytogenetics Core Facility (P30 CA006516). Microarray analyses Breast tumor subtype analyses. Boxplots showing the level of PTK6 mRNA in breast tumor subtypes were derived from three data sets: (1) GSE1992 downloaded from the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/projects/geo/) [48], (2) Rosetta Inpharmatics (http://www.rii.com/ publications/2002/vantveer.html) [49] and (3) a gene expression array (Affymetrix U133) of 129 sporadic primary invasive breast tumors which includes tumors published previously [50], [51]. Boxplots were generated in Matlab and p-values (ANOVA) were calculated in JMP 7.0. Classification of tumors in the Van’t Veer data set was obtained from GEO (GSE4382) [52]. For GSE5460, raw expression values obtained from Affymetrix GENECHIP software were additionally analyzed using DNA- Chip analyzer (dChip) custom software (www.dchip.org). Parallel data of 12 normal breast organoids RNA samples and 7 bulk normal breast tissue specimens were used as normal control. Array probe data were normalized to the mean probe expression level PTK6 Regulates Survival across the cohort; thus, expression levels are relative to a normalized average. Gene filtering and hierarchical cluster analysis were performed using dChip software to identify subtypes of breast tumors using the intrinsic gene list defined by Sorlie et al. [52]. Two PTK6 probes in the array 206482_at and 1553114_a_at were used for analysis of PTK6 transcripts level. The relative levels of hybridization for the two probes were highly correlated (linear regression R = 0.83). Kaplan-Meier curves. For the Van’t Veer data set, normalized PTK6 expression values and time to recurrence data were downloaded from the Rosetta website (http://www.rii.com/ publications/2002/vantveer.html). Samples were divided into three equal tertiles: 98 tumors with highest PTK6 level (.0.053); 99 tumors with intermediate PTK6 level (between 20.021 and 0.056) and 98 tumors with lowest PTK6 level (,20.021). For the ER+ plots, only the 226 samples classified as ER+ by Van’t Veer et al. were used. For the Wang set, PTK6 expression values and time to recurrence data were downloaded from the Gene Expression Omnibus (GEO, GSE2034). Samples were divided into three equal tertiles: 95 tumors with highest PTK6 level (.242.3); 96 tumors with intermediate PTK6 level (between 126.3 and 243.7) and 95 tumors with lowest PTK6 level (,126.9). For the ER+ plots, only the 209 samples classified as ER+ by Wang et al. were used. For all survival curves, the y-axis (probability) is defined as the frequency of relapse-free survival. Kaplan-Meier analyses were carried out using survival package within R language. P-values were derived using the log-rank test and by fitting a Cox proportional hazards regression model. Multivariate Cox propor- tional hazards analysis was performed for PTK6 and clinical prognostic factors including estrogen receptor (ER),HER2 onco- gene, axillary lymph node involvement. Immunoprecipitations, and Western analyses Cells were lysed in RIPA lysis buffer (1%Triton X-100, 1%NaDOC, 0.1%SDS, 20mM Tris pH 7.5, 150 mM NaCl, 1mM EDTA) or RPPA lysis buffer (1% Triton X-100, 50mM HEPES, 150mM NaCl, 1.5mM MgCl2, 1mM EDTA, 100mM NaF, 10mM NaPP, 10% glycerol, 1mM PMSF, 1mM Na3V04) supplemented with protease and phosphatase inhibitors (1mg/ml leupeptin, 1 mg/ml aprotinin, 1 mg/ml pepstatin, 10 mg/ml PMSF, 1 mM NaVO4, 1mM NaF) for 30 minutes at 4uC. Lysates were clarified by centrifugation and supernatants were collected. Proteins were resolved by 8–10% SDS-PAGE gel electrophoresis and immunoblotted using standard techniques. Reverse phase protein array (RPPA) analysis Serially diluted cell lysates were printed on nitrocellulose-coated slides and probed with specific antibodies as previously reported [53]. Signal intensity data were collected and analyzed using software specifically developed for RPPA analyses (http://www. vigenetech.com). The normalized linear values for each sample were divided by the trimmed mean (25%) of the values for that sample. Each value was standardized using a modified z-score. Z~ median of replicates{median of lucif erase replicates s Heatmaps were generated using Cluster 3.0 and Java TreeView 1.1.1. Phase contrast microscopy Cells grown in monolayer cultures, as well as 3D acinar structures were visualized at 20uC using a Nikon TE300 PLoS ONE | www.plosone.org 12 July 2010 | Volume 5 | Issue 7 | e11729