Real-time polymerase chain reaction (PCR) Total RNA extraction from subconfluent cultures of PC-3/Fluc cells and the various clones was performed with RNeasy (Qiagen, Hombrechtikon, Switzerland). Reverse transcription was per- formed with M-MLV-RT (Promega, Wallisellen, Switzerland) and random primers (Roche Diagnostics). Human-specific real-time PCR primers and probes (Applied Biosystems, Rotkreuz, Switzer- land) are listed in Supporting Information (Table S2). Immunoblotting Noggin secretion by PC-3/Fluc cells and by the different clones was determined in concentrated CM, normalized by the total cell number collected from each sample. Proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gels (Mini Protean Gel BioRad) and transferred on Hybond-P membranes (GE Healthcare, Glattburg, Switzerland). Membranes were incubated overnight with 40 ng/ml of a rat monoclonal antibody against the human native noggin protein [20] (RP57-16; kindly provided by Dr. A.N. Economides, Regeneron Pharmaceuticals, Inc.) and detected with a horseradish peroxidase-labeled anti-rat secondary antibody (1:1000; GE Healthcare). Immunoreactivity was visual- ized with the ECL Advanced chemiluminescence substrate (GE Healthcare) using the VersaDoc imaging system (Bio-Rad Laboratories, Reinach, Switzerland) and signal intensity was quantified with QuantityOne imaging software (Bio-Rad Labora- tories). Equivalent protein loading was verified by staining with Coomassie blue the total protein content in the CM. Intra-osseous inoculation of PC-3/Fluc cells, mock and Nog-KD clones The cells were inoculated into the marrow cavity of the left tibia at a concentration of 56105/10 ml of phosphate-buffered saline (PBS), as previously described [18]. Groups of seven animals each were inoculated with either PC-3/Fluc cells, mock or Nog-KD clones. Another group of 7 animals, inoculated with phosphate- buffered saline alone served as control ( = sham). In a first experiment tumor growth was allowed to progress until day 25, at which time animals from all groups were sacrificed because of the severe osteolysis and imminent risk of fracture in the PC-3/Fluc and mock groups. In a further experiment the animals xenografted with the PC-3/Fluc clone were sacrificed at day 23 for the same reason above, while the groups xenografted with Nog-KD 14 and 17 clones were kept for a period of 30 and 33 days, respectively. The xenografted and sham-operated tibiae were dissected and fixed in 4% paraformaldehyde (PFA). Intra-cardiac injection of PC-3/Fluc cells, mock and Nog-KD clones The cells were injected into the left cardiac ventricle at the concentration of 105/100 ml in PBS, as previously described [18]. Groups of ten animals each were injected with either PC-3/Fluc or mock 4 or Nog-KD 17 clones. Animals were sacrificed 28 days after injection. Radiography Radiographs of the mice were taken weekly using a Faxitron radiography system (MX-20, Faxitron X-Ray Corporation, Edimex, Le Plessis, France) to monitor changes in bone structure and radiodensity. Role of Noggin in Osteolytic Bone Metastasis previously described [18] using an ultrasensitive charge-coupled device (CCD) camera (NightOWL LB, Berthold Technologies, Bad Wilbad, Germany). In vivo photon counts were normalized for the in vitro luciferase activity by PC-3/Fluc clone, mock and Nog- KD clones. Three-dimensional micro-computer tomography (m-CT) of bone lesions To obtain qualitative and quantitative assessment of the tumor- induced modifications of the bone structure, sham and tumor- bearing tibiae were scanned ex vivo, at the end of the experimental period, with a m-CT40 scanner (SCANCO Medical AG, Bru¨ ttisellen, Switzerland) at a resolution of 6 mm. The region of interest was selected from the scout view and 1600 micro- tomographic slices were acquired, covering three quarters of the length of the tibia. Tridimensional images were obtained through reconstruction of cross sectional images. Quantitative analysis of primary parameters such as total volume (TV) and bone volume (BV) was performed on a volume of interest equivalent to 166 slides (1 mm in length) at approximately 2.5 mm distal from the cleft of the knee joint as reference point. Peripheral quantitative computed tomography (pQCT) Bone mineral content in the xenografted tibiae was determined ex vivo with a small animal pQCT scanner (XCT Research SA; Norland Stratec, Pforzheim, Germany) at the end of the experimental period. Measurements were performed at 2 and 3 mm distal from the reference point (cleft of the knee joint). Bone histomorphometry Fixed tibiae were decalcified in a solution of PFA 0.5%/EDTA 15% for 20 days and processed for paraffin embedding. Four-mm sections were stained for tartrate-resistant acid phosphatase (TRAP) as described previously [21], counterstained with hematoxylin and examined by light microscopy. Osteoclasts were counted as multinucleated TRAP-positive cells on the endosteal surface of cortical and trabecular bone in sham-operated tibiae, and on the residual bone adjacent to tumor cells in cancer cell- xenografted tibiae. The number of osteoclasts (N.Oc/BS; /mm) and of active osteoblasts (N.Ob/BS; /mm), the bone surface covered by osteoclasts (Oc.S/BS; %) and osteoblasts (Ob.S/BS; %) were determined using the OsteoMeasure histomorphometry software (OsteoMetrics Inc., Decatur, GA, USA). Osteoclasts and osteoblasts were counted in randomly selected fields of an area .1 mm2, approximately at 1.5 mm from the growth plate. Bone parameters were expressed as recommended in the report of the ASBMR Histomorphometry nomenclature committee [22]. Statistical analysis Graph Pad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses. The parametric one-way ANOVA test with a Bonferroni post-test was used to analyze the RNA expression data, the bone architecture parameters obtained by m-CT and pQCT, and the histomorphometric data. To compare the cell proliferation rate in vitro and in vivo the parametric two-way ANOVA test was employed. P values smaller than or equal to 0.05 were considered as significant. Supporting Information In vivo bioluminescent imaging (BLI) Tumor growth was monitored non-invasively in the living animals by bioluminescent imaging (BLI) at weekly intervals as Figure S1 Luciferase expression does not modify the osteolytic potential of PC-3 cells and moderately affects their gene expression in vitro. A. Representative images of PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e16078