scratching with a sterile pipette tip as described elsewhere [87]. For invasion assay, 100,000 cells per well were plated in a xCelligence CIM-culture plate array. Blocking PTN antibody, recombinant PTN protein (R&D) and Glycine/Tris-HCl pH 7.5 antibody elution buffer (Control) were used as chemo attractant/ repellent. Impedance was measured for the first 6 hours of cell invasion with xCelligence Instrument (Roche). Mammosphere Three-dimensional (3D) Culture 3D culture was performed as described in [50]. Briefly, cells were resuspended in HAM’s F12 supplemented media containing EGF at 20 ng/ml, 1.25% FBS, 2% Matrigel (BD Bioscience) and either blocking PTN antibody alone or in combination with recombinant PTN protein (R&D) or Glycine/Tris-HCl pH 7.5 antibody elution buffer (Control). 400 ml of the cells/media mix was added per well in an 8 well Nunc Lab-Tek II Chamber Slide System precoated with Matrigel. Cells were seeded at 8,000 cells/ well. Media containing either the antibody or the antibody with equimolar recombinant PTN protein or the elution buffer was replenished every two days. Pictures of the wells were taken at day 1, 3 and 5. Immunofluorescence Primary mammary epithelial cells passage #3 grown in poly-D- lysine coated 8 well Lab-Tek II Chamber Slide System (Nunc) were fixed and permeabilized twice with 4% formaldehyde, 0.2% Triton X in cold PBS for 10 minutes at room temperature, washed with ice-cold PBS, blocked with 1% BSA in PBS and incubated with a goat anti-human PTN (1:100; R&D) antibody for 1h at room temperature. After three washings with PBS, cells were incubated with Alexa Fluor 488 anti -goat IgG (1:1000; Invitrogen) and with Alexa Fluor 568 Phalloidin (1:50; Invitrogen) for 1h at room temperature. After three washings with PBS, cover slips were mounted with ProLong Gold Antifade with DAPI (Invitrogen) and fluorescence was analyzed on an Olympus Fluoview FV300 Laser Confocal Microscope (Olympus, Hamburg, Germany). Immunostaining of mammospheres was performed as described in [80]. Briefly, Matrigel was dissolved with PBS-EDTA and mammosphere suspensions were transferred to poly-D-lysine coated slides. Mammospheres were fixed in 4% formaldehyde References 1. Schulte AM, Wellstein A (1997) Pleiotrophin and related molecules. In: Bicknell R, Lewis CM, Ferrara N, editors. Tumour Angiogenesis. Oxford, New York, Tokyo: Oxford University Press. 273–289. 2. Li YS, Milner PG, Chauhan AK, Watson MA, Hoffman RM, et al. (1990) Cloning and expression of a developmentally regulated protein that induces mitogenic and neurite outgrowth activity. Science 250: 1690–1694. 3. Iwahara T, Fujimoto J, Wen D, Cupples R, Bucay N, et al. 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Spheres were stained using as primary antibody ki67, Caspase 3 mouse anti- human laminin c2 (D4B5 clone; 1:200; Millipore) and goat anti- human PTN (1:100; R&D) diluted in blocking buffer and incubated with Alexa Fluor 488 anti-mouse IgG (H+L) (1:1000; Invitrogen). Cover slips were mounted as above. Statistical and Image Analysis Prism 5 for Mac (GraphPad, San Diego, CA) was used for all statistical analyses. Adobe Photoshop CS3 was used to process the pictures for immunohistochemistry, whole mounts, Western blot, mammosphere three-dimensional culture and immunofluores- cence. Brightness/Contrasts and Levels adjustments were applied to the whole image. All images were adjusted to the same level. Image J was used to measure the ductal length, terminal end buds and terminal ends diameter, and mammosphere surface area. Immunofluorescence images were overlaid using the Metamorph Software. This article contains supporting information. Supporting Information Figure S1 Anti-PTN antibody binding activity to PTN and its detection from mouse serum of treated mice. (A) Anti-PTN antibody binding to immobilized PTN by ELISA. Anti- PTN antibody KD-value = 1 nM. Control = antibody elution buffer. OD defines absorbance at 450 nm. (B) PTN blocking antibody detection from mouse serum of treated mice (anti-PTN; n = 9 mice, grey) versus control (n = 5 mice, white) by PTN immobilized ELISA. Absorbance at 450 nm is defined as arb.u. Data are means 6 SE; ***P = 0.0004; two-tailed student-t test. (TIF) Author Contributions Performed the experiments: SMR ETB SCM KAG VO RTH. Analyzed the data: SMR ETB SCM ATR AW. Wrote the paper: SMR ETB ATR AW. GAM (pleiotrophin) is associated with epithelial-mesenchymal interactions during fetal development and organogenesis. Development 121: 37–51. 10. Sakurai H, Bush KT, Nigam SK (2001) Identification of pleiotrophin as a mesenchymal factor involved in ureteric bud branching morphogenesis. 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