microscope equipped with a CCD camera, using a 46/0.13 objective. Images were acquired using ImageJ software, converted to TIFF images and arranged using Adobe Photoshop 7.0. Immunofluorescence analyses and confocal microscopy Acinar structures were fixed in 2% formalin (Sigma) at room temperature for 20 minutes and permeabilized in 0.5% Triton X- 100 in PBS for 10 min at 4uC. Immunostaining of acinar structures was carried out as previously described [47] and imaged at 20uC. DAPI was purchased from Molecular Probes. Confocal analyses were performed using the Nikon TE2000 microscope with the C1plus confocal microscope system equipped with krypton-argon (488 line) and HeNe (543 and 633 lines) lasers. Structures were analyzed with a 406/1.3 objective and images were acquired using the Nikon C1 Confocal software. All images were converted to TIFF format and arranged using Adobe Photoshop 7.0. Supporting Information Figure S1 siRNAs targeting PTK6 identified by the screen inhibit anchorage-independent survival of IGF-1R cells. A) Parental and IGF-1R overexpressing MCF-10A cells transfected with control or PTK6 siRNA (OTP, Dharmacon) were cultured in suspension for 48 hours. Lysates were probed with the indicated anti-sera. B) Western analysis demonstrates down regulation of PTK6 expression in IGF-1R cells by siRNAs targeting PTK6 identified by the screen to induce changes in viability. IGF-1R cells transfected with multiple PTK6 siRNAs, including a pool of siRNAs from a distinct vendor (OTP, Dharmacon), were cultured in suspension for 48 hours. Lysates were prepared and probed with the indicated anti-sera. C) siRNAs targeting PTK6 inhibit anchorage-independent survival of IGF-1R cells. IGF-1R cells transfected with multiple PTK6 siRNAs, including a pool of siRNAs from a distinct vendor (OTP, Dharmacon), were cultured in suspension for 48 hours. Cell death was assessed as described in Figure 1. The average fold change from three independent experiments is shown. Error bars indicate standard deviation. Found at: doi:10.1371/journal.pone.0011729.s001 (0.24 MB TIF) Figure S2 Overexpression of PTK6 alone in HMEC does not enhance growth in soft agar. Immortalized human mammary epithelial cells (HMLE) overexpressing vector control, F-PTK6 or MF-PTK6, were seeded in soft-agar, and colony formation was assessed after four weeks. A representative experiment of three replicate experiments using triplicate wells for each cell line is shown. Found at: doi:10.1371/journal.pone.0011729.s002 (0.08 MB TIF) Figure S3 Expression of PTK6 in breast and ovarian cancer cell lines. A) Western blot analysis was performed to assess levels of References 1. Chiarugi P, Giannoni E (2008) Anoikis: a necessary death program for anchorage- dependent cells. Biochem Pharmacol 76: 1352–1364. 2. Simpson CD, Anyiwe K, Schimmer AD (2008) Anoikis resistance and tumor metastasis. Cancer Lett 272: 177–185. 3. Martin SS, Ridgeway AG, Pinkas J, Lu Y, Reginato MJ, et al. (2004) A cytoskeleton-based functional genetic screen identifies Bcl-xL as an enhancer of metastasis, but not primary tumor growth. Oncogene 23: 4641–4645. 4. Douma S, Van Laar T, Zevenhoven J, Meuwissen R, Van Garderen E, et al. (2004) Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB. Nature 430: 1034–1039. 5. Mawji IA, Simpson CD, Gronda M, Williams MA, Hurren R, et al. (2007) A chemical screen identifies anisomycin as an anoikis sensitizer that functions by decreasing FLIP protein synthesis. Cancer Res 67: 8307–8315. 6. Simpson CD, Mawji IA, Anyiwe K, Williams MA, Wang X, et al. (2009) Inhibition of the sodium potassium adenosine triphosphatase pump sensitizes cancer cells to anoikis and prevents distant tumor formation. Cancer Res 69: 2739–2747. PTK6 Regulates Survival PTK6 protein expression in a panel of breast cancer cell lines representative of different subtypes, as classified in Neve et al. [54]. B) Western blot analyses were performed to assess levels of PTK6 expression in human ovarian surface epithelial cells and ovarian cancer cell lines. Found at: doi:10.1371/journal.pone.0011729.s003 (0.53 MB TIF) Figure S4 Effect of IGF-1R kinase inhibitor on IGF-1R signaling. IGF-1R overexpressing MCF-10A cells were treated in suspension cultures containing IGF-1 (100ng/ml) for 48 hours with the indicated concentrations of IGF-1 receptor kinase inhibitor (BMS-536924; Selleck). Lysates were probed with the indicated antisera to assess IGF-1R autophosphorylation and phosphorylation of Akt. Found at: doi:10.1371/journal.pone.0011729.s004 (0.28 MB TIF) Figure S5 Cluster analysis of chromosome 20 copy number alterations in breast cancers. The heatmap is based on SNP array copy number data from 93 breast tumor samples with 511 chromosome 20 genes (ordered from p arm to q arm). Only data above log2 value 0.5 or below 20.5 were used (representing copy numbers above 2.8 or below 1.4). Left, cluster of the entire chromosome 20; right, magnification of the yellow box. Dark blue, red, and light blue colors represent losses, gains, and no significant change in copy number. Found at: doi:10.1371/journal.pone.0011729.s005 (1.07 MB TIF) Figure S6 Higher levels of PTK6 mRNA are associated with clinical measures of poor prognosis in multiple breast cancer data sets. Box plots and data are from the Oncomine database (www. oncomine.org). Background corrected expression data are log2 transformed and the median value per microarray is scaled to zero by subtracting the median from each value. The standard deviation of values for each microarray is scaled to 1. P-values are derived from the Student’s t-test. 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