K18 (Reverse) TCCAAGTTGATGTTCTGGTTTTT- CATGG ALK (Forward) GGACGGGACACAGCTCCATG ALK (Reverse) GCACTCCAGACCATATCGACTGCG CD10 (Forward) CTCTCTGTGCTTGTCTTGCTC CD10 (Reverse) GACGTTGCGTTTCAACCAGC CD29 (Forward) ATGCCAAATCTTGCGGAGAAT CD29 (Reverse) TTTGCTGCGATTGGTGACATT CD49f (Forward) TGCAGAGGGCGAACAGAAC CD49f (Reverse) GCACACGTCACCACTTTGC SCA-1 (Forward) AGGAGGCAGCAGTTATTGTGG SCA-1 (Reverse) CGTTGACCTTAGTACCCAGGA Primers for Vimentin were described in [81]. Immunohistochemistry Abdominal mammary glands were fixed in formaldehyde and embedded in paraffin. Immunohistochemical analysis of 5 mm section was done using the Vectastain ABC Elite System (Vector Labs). The primary goat anti-human PTN antibody (R&D) diluted 1:500 was incubated at 4uC overnight. The slides were counter- stained with hematoxylin (Sigma Aldrich). Tissue sections are representative of 3–4 mice. Image capturing for stained section was performed with a Nikon E600 Epifluorescence Microscope. Whole Mount Mammary gland #4 was excised from 6-week-old mice whole mount stained [82] and ductal penetration and branching points were quantified as described in [83]. Ductal penetration was expressed as the average length (mm) of the three longest epithelial ducts in each mammary gland. Branch points of the measured ducts were counted and divided by the average duct length (mm) in each mammary gland. Terminal End Buds (TEBs) were defined as having a largest diameter $100 mm [54]. Terminal Ends (TEs) were defined as having a largest diameter ,100 mm. This included terminal ducts and alveolar buds. TEB and TE numbers are given per duct. The percentage of TEBs was calculated relative to the total number of ductal ends, i.e. TEBs plus TEs [55]. Anti-PTN Antibody Production, Administration and Detection The hybridoma cell line producing the anti-PTN mouse monoclonal antibody (clone 3B10) was previously described [84] and was cultured in DMEM (Invitrogen) media supplemented with 10% fetal bovine serum in a CELLine Device (BD Biosciences). Media containing the antibody was collected every 7 days, pooled and purified using a 5 ml HiTrap Protein G HP Column (GE Healthcare). Antibody concentration was assessed against a bovine serum albumin standard after SDS-PAGE and Coomassie staining. Antibody binding to PTN was titrated using an ELISA with immobilized PTN and yielded a KD-value of 1 nM. FVB mice (3 weeks old) were injected intraperitoneally with a total dose of 2 mg/kg of the antibody or with PBS as a control. Serum samples from control and treated 6-week-old FVB mice were diluted 1:100 in PBS and tested in an ELISA assay with immobilized recombinant human Pleiotrophin (PTN; at 25 ng/ well; R&D Maxisorb Nunc plates). 5% (w/v) non-fat dry milk was used to block non-specific binding. Superfluous solution was washed off with 3 washes with 0.05% Tween20 (Fisher Scientific) in PBS ( = PBST). Diluted serum samples were incubated for 1 h at room temperature. Anti-PTN antibody stock solution diluted to 100 nM was used for calibration. Wells were washed 5 times with PBST. A horseradish peroxidase-linked sheep, anti-mouse IgG PTN in the Mammary Gland was the secondary antibody (Amersham Biosciences, GE Health- care), 1-Step Turbo TMB ELISA Substrate (Thermo Scientific) the detection reagent and sulfuric acid the stop solution. The absorbance was read at 450 nm. Western Blot and Phosphorylation Studies Whole mammary glands #3 from 6-week-old FVB mice was harvested and lysates (5 mg/well) were analyzed either using a MS6000 MAP Kinase Whole Cell Lysate kit (Phospho protein) and MS6000 MAP Kinase (Total Protein) Whole Cell Lysate kit (Meso Scale Discovery) according to manufacturer’s instructions. Whole mammary gland lysates and whole cell lysates from primary cultures were run (75 mg/lane) on a 4–20% Tris-Glycine NOVEX gel (Invitrogen), transferred to PVDF membranes. Primary epithelial cells were harvested 24 hours after treatment with UO126 (500 nM) alone or in combination with anti-PTN blocking antibody. Immunoblot studies for phospho-MAPKs and total MAPKs were performed with the respective rabbit polyclonal antibodies (1:1000 dilution; Cell Signaling). Visualization was performed using Immobilon Western HRP substrate Peroxide Solution (Millipore) with horseradish peroxidase-linked sheep anti- rabbit immunoglobulin G as a secondary antibody (Amersham Biosciences, GE Healthcare). Primary Mammary Epithelial and Fibroblast Cell Harvest and Culture Mammary glands #3 and #4 were dissected from 7 to 9-week- old FVB mice carefully removing muscles, lymph nodes and nipples. Glands were minced and digested in 1 mg/ml collagenase (Sigma-Aldrich) solution for 90 minutes at 37uC on a shaker at 150 rpm. The digested tissue was centrifuged for 10 minutes at 6006g and washed 4 times. DMEM/F12 (Invitrogen) media supplemented with Penicillin/Streptomicin (Invitrogen) 1X and 50 mg/ml Gentamicin (Invitrogen) was used to prepare the collagenase solution and to wash the digested tissues. Cells were resuspended and plated on collagen-coated plates (Costar) in MEC growth media (Ham’s F12 (Invitrogen) supplemented media with 10% fetal bovine serum, 16 mg/ml insulin (Invitrogen), 10 ng/ml EGF (Roche Applied Biosciences), 1 mg/ml hydrocortisone (Sigma-Aldrich), 50 mg/ml gentamicin (Invitrogen), 4 ng/ml cholera toxin (Sigma-Aldrich) and 1X penicillin/streptomycin). To avoid fibroblast contamination, cells in the supernatant were transferred to uncoated plates 20 minutes after initial plating. Fibroblasts growing on collagen coated plates (BD Biosciences) and epithelial cells were incubated at 37uC and 5%CO2 and cultured up to passage #3 and then used for RNA extraction, immunofluorescence, three-dimensional culture, real-time imped- ance sensing and Western blot analysis. Cell Proliferation, Migration and Invasion Assay: Electric Impedance Sensing Primary epithelial cells were plated on microelectrodes embed- ded wells and the impedance of the cell monolayer was monitored in real time as described elsewhere [85,86]. For proliferation and migration assay, 8-10,000 cells per well were plated in a xCelligence E-culture plate array (Roche Applied Biosciences) and followed until confluence and steady state impedance. For proliferation assay, cells were stimulated 20 hours after plating with either blocking PTN antibody alone or in combination with recombinant PTN protein (R&D) or Glycine/Tris-HCl pH 7.5 antibody elution buffer or bFGF (Invitrogen) and cell monolayer impedance was monitored until confluence. For migration assay, cell monolayers were allowed to form and were disrupted by PLOS ONE | www.plosone.org 11 October 2012 | Volume 7 | Issue 10 | e47876