TGF-b Inhibition in Myeloma inoculation of INA6 cells and serum levels of the soluble human IL-6 receptor were measured as described in Materials and methods. **, ,0.01. B. X- ray photographs of the rabbit bones were taken before and 6 weeks after the treatment. Representative images are shown. C. At 6 weeks after the treatment, the rabbit bones were removed. The samples were sectioned and stained with H&E. Representative specimens of control and treated rabbit bones are shown in the upper and lower panels, respectively. Original magnifications were 640 and 6200 as indicated. D. Histomorphometrical analyses of the implanted rabbit bone sections. BV/ TV, Ob.S/BS and tumor/TV were measured in decalcified implanted rabbit bone sections stained with H&E as described in Materials and Methods. Data are expressed as means +/2 SD. *, p,0.05. E. MM cell lines, INA6, RPMI8226, U266 and KMS-12 at 56104/mL, were cultured in the absence or presence of Ki26894 (10 mM) or SB431542 (3 mM). After culturing for 3 days, viable MM cell numbers were counted. Percent changes from the control are shown. Results are expressed as means +/2 SD. doi:10.1371/journal.pone.0009870.g007 Transfection MC3T3-E1 cells were seeded in 6-well culture plates at 50% confluence and co-transfected with 2 mg of chimeric firefly luciferase reporter plasmid with Renilla luciferase reporter plasmid (pRL-TK; Promega, Madison, WI) as an internal control using GenePorter2 transfection reagent (Genlantis Inc., San Diego, CA) in opti-MEM (Invitrogen, Carlsbad, CA) supplemented with 1% FBS. A TCF/LEF binding site reporter plasmid was used (TopflashTM; Upstate Biotechnology, Lake Placid, NY). The medium was replaced with aMEM containing 1% FBS at 16 hours after transfection. For dual-luciferase assays, the cells were washed and lysed with a passive cell lysis buffer (Promega, Madison, WI), and both firefly and renilla luciferase activity was measured with a luminometer (ATTO, Tokyo, Japan) by mixing the luciferase substrate (Promega) with 10 mL of cell lysate. Transcriptional activity was expressed as renilla luciferase activity. Western blot analysis Cells were collected and lysed in a lysis buffer (Cell Signaling) supplemented with 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail solution (Sigma). Cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% non-fat dry milk in TBS with 0.01% Tween 20 (MP Biomedicals, Irvine, CA) for one hour at room temperature and incubated for 16 hours at 4uC with anti-phospholylated-Smad1, anti-Smad1, or anti-b-actin antibody. After washing, secondary horseradish peroxidase–conjugated antibody was added, and the membranes were developed using the Enhanced Chemilumines- cence Plus Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ). Cell viability and cell cycle analysis Viable cell numbers were measured by a cell proliferation assay using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo- phenyl)-2H-tetrazolium (WST-8; Kishida Chemical, Osaka, Japan), or a trypan blue exclusion assay, as previously described.[12] Apoptosis was evaluated by staining cells with an annexin V-FITC and propidium iodide (PI) labeling kit (MEB- CYTO Apoptosis Kit; MBL,Nagano, Japan) according to instructions. Cell cycle distribution was evaluated by staining cells with a bromodeoxyuridine (BrdU) and 7-amino-actinomycin D (7- AAD) labeling kit (BrdU Flow Kit; BD Pharmingen, San Diego, CA) according to instructions. Briefly, cells were cultured with BrdU at 10 mM for the last 2 hours of culture. They were collected, fixed, and incubated with a FITC-conjugated monoclo- nal antibody against BrdU. 7-AAD (20 mL per sample) was added for DNA staining. BrdU incorporation and DNA content were analyzed by flow cytometry. References MM mouse model and measurement of serum soluble human IL-6 receptor levels The SCID-rab MM mouse model was prepared as de- scribed.[28] Briefly, femora and tibiae from 4-week-old Japanese white rabbits (Kitayama Labes, Nagano, Japan) were implanted subcutaneously into six-week-old male CB-17 SCID mice (CLEA Japan, Tokyo, Japan). After allowing bone engraftment for 4 weeks following the implantation, 16106 INA-6 cells in 50 mL of phosphate-buffered saline (PBS) was inoculated directly into the bone marrow cavity of the rabbit bones. A TGF-beta type I receptor kinase inhibitor, Ki26894, was mixed with food (0.08%) and administered orally via chow as previously reported.[29] The treatment with Ki26894 was initiated 1 day after the inoculation of INA6 cells. Serum levels of soluble human IL-6 receptor derived from INA6 cells were used as a marker for tumor burden as previously described.[27] Mouse sera were serially collected and serum levels of soluble human IL-6 receptor were measured with an enzyme-linked immunosorbent assay (human IL-6 sR; R&D Systems, Minneapolis, MN). Six weeks after the MM cell inoculation, the mice were anesthetized with an i.p. injection of pentobarbital (Dainippon Pharma, Osaka, Japan). X-ray photo- graphs of the rabbit bones were taken, and the rabbit bones were collected. The rabbit bones were fixed in 10% phosphate-buffered formalin, and decalcified with 10% EDTA. The samples were further embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin (H&E) for histopathologic examination. Bone volume per tissue volume (BV/TV), osteoblast surface per bone surface (Ob.S/BS) and tumor per tissue volume (tumor/TV) were measured in decalcified implanted rabbit bone sections stained with H&E as the indices of bone destruction, osteoblast function and a tumor burden, respectively, as described previously. [48,49] Quantification of data Raw image data of mineralized nodule formation and immunobolotting were quantified with the Multi Gauge version 3.0 software (Fujifilm, Tokyo, Japan). Statistical analysis Statistical significance was determined by a one-way analysis of variance (ANOVA) with Scheffe post hoc tests. The minimal level of significance was a p value equal to 0.05. Author Contributions Conceived and designed the experiments: KT MA SK AN KY SO TM. Performed the experiments: KT MA MH AO HA SK TH OT HM SN AN KK KY SO. Analyzed the data: KT MA MH AO HA SK TH AN KY SO TM. Contributed reagents/materials/analysis tools: MH AO HA SK OT HM SN AN KK KY TM. Wrote the paper: KT MA KY TM. 1. Abe M, Hiura K, Wilde J, Moriyama K, Hashimoto T, et al. (2002) Role for macrophage inflammatory protein (MIP)-1alpha and MIP-1beta in the development of osteolytic lesions in multiple myeloma. Blood 100: 2195–2202. 2. Hashimoto T, Abe M, Oshima T, Shibata H, Ozaki S, et al. (2004) Ability of myeloma cells to secrete macrophage inflammatory protein (MIP)-1alpha and MIP-1beta correlates with lytic bone lesions in patients with multiple myeloma. Br J Haematol 125: 38–41. PLoS ONE | www.plosone.org 13 March 2010 | Volume 5 | Issue 3 | e9870