the specific Wnt isoforms involved have not been identified, although exogenous Wnt3a treatment can increase cranial osteoblast differentiation and enhance bone mineralization [33,34]. Interestingly, the activities of the LRP6 antibodies on endogenous Wnt signaling and biological processes can be used to predict a subset of Wnt isoforms active in specific models and systems. RNAi of candidate Wnts identifies Wnt1, Wnt2, Wnt3, and Wnt7b as the specific isoforms mediating autocrine Wnt signaling in tumor cell lines M14 (melanoma), NCI-H23 (NSCLC), PA-1 (teratocarcinoma), and Hs578T (breast), respec- tively. While mutations in Wnt genes have been linked to seve- ral human diseases (http://www.stanford.edu/,rnusse/diseases/ Humangeneticdis.htm), further identification of specific Wnt isoforms mediating tumorigenesis in different cancer types, skeletal diseases, and stem cell maintenance will clearly be critical for realizing the therapeutic potential of Wnt coreceptor antibodies. With this knowledge, LRP5/6 antibodies and their alternative formats, including one-armed and bipecific antibodies, may enable finely tuned antagonism and potentiation of the many tissue- specific roles that the 19 human Wnt isoforms have evolved to perform. Materials and Methods Ethics Statement All experiments using mice were approve by the Genentech Institutional Animal Care and Use Committee (protocol numbers 07-0194G, 09-1648, and TH10-0841). Cell Culture and Cell Assays Cell lines EKVX and M14 were obtained from National Cancer Institute (National Institutes of Health, Frederick, MD) and grown in RPMI-1640 medium supplemented with 10% fetal bovine serum and 2 mM glutamine. JHH-1 cells were obtained from Japan Health Science Foundation (Health Science Research Resources Bank, Osaka, Japan) and grown in Williams’ Medium E with the same supplements. All other cell lines were purchased from American Type Culture Collection (ATCC) and maintained as recommended. Luciferase reporter assays were performed as previously described [21] using TOPglow (Upstate) or TOPbrite [36] firefly luciferase Wnt reporter, or pRL-SV40 Renilla luciferase (Pro- mega) plasmid. Cells were treated with antibodies for 16–20 h, starting 24 h after transfection. Either purified Wnt3a protein was added to cells starting 1 h after initiating antibody treatment, or Wnt expression constructs were co-transfected with luciferase reporters. Firefly luciferase levels were normalized for transfection efficiency to Renilla luciferase levels, and the relative luciferase units (RLU) were additionally normalized to the level in cells not stimulated with Wnt. HEK293 and Hs578T cell lines stably integrated with TOPbrite reporter were selected for hygromycin resistance. Expression of Wnt luciferase reporter was normalized to cell number based on stably integrated SV40-driven Renilla luciferase for HEK293 cells or on the MultiTox-Fluor cell viability assay (AFC fluorescence; Promega) for Hs578T cells. For quantitative real-time PCR (qPCR) expression analysis, RNA was isolated from cells using the RNeasy kit (QIAGEN), and reactions were performed with the TaqMan One-Step RT-PCR Master Mix Reagents Kit (Applied Biosystems) on the 7900 HT Fast Real-Time PCR System (Applied Biosystems). Relative RNA levels were calculated using the DDCt method and normalized to human GAPDH or mouse Rpl19 RNA levels within the same sample, and additionally normalized to samples from cells with no Wnts Specify LRP6 mAb Activity addition (NA) of Wnt3a, antibody, or other proteins. The primer and probe sets, listed 59 to 39 for forward primer, reverse primer, and probe sequences, respectively, are SP5: AATGCTGCT- GAACTGAATAGAAA, AACCGGTCCTAGCGAAAA, CCG- AGCACTGTTTCAAATCTCCCA; ZNRF3: TGAGAGTGT- GACATTGTTGGAA, GTAAAATCTGTGTGCAATTATCA- TGT, AATCATTGAAAATGACTAACACAAGACCCTGTA- AAT; mouse Mmp7: TGAGGACGCAGGAGTGAA, CCCAG- AGAGTGGCCAAAT, CCTGTTTGCTGCCACCCATGA. Prim- ers and probes used for human APCDD1, AXIN2, GAD1, LEFTY2, and SAX1, and for mouse Rpl19 and Axin2, were previously described [21,37]. GAPDH primers and probe were purchased from Applied Biosystems. For reporter gene and qPCR assays, all figures represent the mean and standard deviation of three or four experimental replicates. For Western analysis, 1.26106 HEK293 cells were seeded onto 10-cm dishes and treated 3 days later with 10 mg/ml antibody, 2.5 mg/ml DKK1 (R&D Systems), or 2.5 mg/ml Fzd8CRD-Fc protein [21] for 1 h before adding 0.2 mg/ml Wnt3a protein for an additional 1 h. Cells were washed twice with cold PBS and lysed in 0.5 ml SJC lysis buffer [36] on ice. For sequential affinity purification of LRP6 prior to Western analysis, HEK293 cells were transfected with 0.33 mg/ml each of LRP6-flag and LRP6- HA expression constructs using FuGENE 6 reagent (Roche), treated 1 day later with 1 mg/ml antibody for 1 h on ice, washed with PBS, and lysed in SJC buffer containing 1% n-dodecyl-b-D- maltoside (DDM). Cold cell lysate was homogenized by five passes through a 28-gauge needle and clarified by centrifugation at 25,000 x g for 15 min. EZview Red ANTI-FLAG M2 affinity gel (60 ml settled resin; Sigma-Aldrich) was added to 500 mg of lysate protein in 500 ml lysis buffer and rotated overnight at 4uC. Affinity gel was washed twice in lysis buffer, twice in TBS (10 mM Tris HCl, 150 mM NaCl, pH 7.4) containing 1% DDM, and subjected to two sequential affinity elutions, each using 300 ml of TBS containing 1% DDM and 200 mg/ml FLAG peptide (Sigma- Aldrich) for 30 min at 4uC. Pooled eluate was combined and incubated with nProtein A Sepharose and Protein G Sepharose Fast Flow (25 ml of a 1:1 mix of settled resins; GE Healthcare) overnight at 4uC. Sepharose was washed three times with TBS containing 1% DDM, once with TBS, and incubated at 90uC for 10 min in 60 ml NuPAGE LDS sample buffer and sample reducing agent (Invitrogen). 20 mg of protein, or 20 ml of affinity purified sample, was electrophoretically resolved on a denaturing SDS-polyacrylamide gel (4–12%), transferred to nitrocellulose membrane, and probed with antibodies against phospho- and total LRP6 (Cell Signaling Technology), b-catenin (BD Transduction Laboratories), b-actin (Sigma-Aldrich), GAPDH (Millipore), HA (Covance), and FLAG (Sigma-Aldrich). Proteins were visualized using infrared labeled secondary antibodies (Rockland Immuno- chemicals) and Odyssey imager (LI-COR). For RNAi experiments, ON-TARGETplus double-stranded siRNA oligomers (Thermo Scientific) were resuspended and used for 72-hour transfection according to the manufacturer’s instruc- tions with DharmaFECT 2 reagent at a final dilution of 1:500, individual siRNAs at 20 nM, and pools of four siRNAs at 80 nM. Individual control siRNAs used for LRP5, LRP6, and non- targeting were Thermo Scientific catalog numbers J-003844-06, J- 003845-11, and D-001810-01-05, respectively. For fluorescence-activated cell sorting (FACS), HEK293 cells stably integrated for LRP6 expression were harvested in PBS, washed with PBS containing 5 mM EDTA and 1% fetal bovine serum, and incubated for 1 h on ice with 0.5 mg/ml primary antibody, with or without pre-incubation with purified LRP6 E1- E4-His protein for 1 h, in 96-well format at 500,000 cells per well. PLoS ONE | www.plosone.org 14 September 2010 | Volume 5 | Issue 9 | e12682