forced expression of EGR1 inhibited osteosarcoma cell invasion by down-regulation of urokinase plasminogen activator (uPA) and urokinase receptor (uPAR). Materials and Methods Cell culture 143B, Saos-2, HOS, and MG63 cells were purchased from the American Type Culture Collection (ATCC, USA). NOS-1 was provided by the RIKEN BRC through the National Bio-Resource Project of The MEXT, Japan (Tsukuba, Japan) [12]. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supple- mented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml). Human osteoblast cells (NHOst) were purchased from Sanko Junyaku (Tokyo, Japan). NHOst was cultured with OBMTM (Cambrex, East Rutherford, NJ, USA) or DMEM supplemented with 10% FBS. All cells were grown in a humidified atmosphere containing 5% CO2 at 37uC. Anti-tumor agents Doxorubicin, methotrexate, and etoposide were purchased from Sigma-Aldrich (MO, USA). Cisplatin was purchased from LKT laboratories (MN, USA). Patient’ specimens All human osteosarcoma biopsy specimens were obtained from primary lesions. Biopsy was performed before chemotherapy or radio therapy to make the diagnosis. Normal bone tissue was obtained from femur during total hip arthroplasty. Specimens of OS6, OS8, and OS9 tumors were obtained during tumor resection in osteosarcoma patients who received chemotherapy. Doxrubicin, methotrexate, and cisplatin were given to these three patients according to COSS-86 protocol. We compared the EGR1 expressions in the biopsy specimens and the resected tumor specimens obtained from these patients. The study protocol was approved by the institutional review board of the Kagoshima University. All patients and controls gave written informed consent. Real-time PCR For real-time PCR, total RNA was obtained 24 h, 48 h, and 5 days following drug treatment. DNase-treated and reverse- transcribed using oligo(dT) primers as described by the manufac- turer (Invitrogen, Carlsbad, CA, USA). Reactions were run using SYBR Green (BIO-RAD, Hercules, CA, USA) on a MiniOpti- conTM machine (BIO-RAD). The comparative Ct (DDCt) method was used to determine fold change in expression using GAPDH or ACTB. Each sample was run minimally at three concentrations in triplicate. All primer sets amplified 150- to 200-bp fragments. The primers sequences used were follows: for EGR1: 5-CAG- CACCTTCAACCCTCAG-3, 5- CACAAGGTGTTGCCACT- GTT-3; uPA: 5- TGTGAGATCACTGGCTTTGG-3, 5- GTCA- GCAGCACACAGCATTT-3; uPAR: 5- TGAAGAACAGTGC- CTGGATG-3, 5- TGTTGCAGCATTTCAGGAAG-3; GAPDH: 5- GAAGGTGAAGGTCGGAGTC-3, 5- GAAGATGGTGAT- GGGATTTC-3; ACTB: 5-AGAAAATCTGGCACCACACC-3, 5-AGAGGCGTACAGGGATAGCA-3. MTT assay Following 100 ng–1 mg cisplatin, 1 ng–10 ng methotrexate, 50 ng–1000 ng etoposide, or 10 ng–100 ng doxorubicin treat- ment, we performed MTT assay to evaluate the osteosarcoma growth as previously reported [13]. In addition, we transfected control vector or EGR1 expression vector, and examined Chemotherapy Prevents OS Invasion by EGR1 osteosarcoma cell growth by MTT assay. Cells were incubated with substrate for MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide) for 4 hours, and washed with PBS and lysed to release formazan from cells. Then cells were analyzed in a Safire microplate reader (BIO-RAD) at 562 nm. Vector transfection EGR1 expression vector was purchased from Origene (Mary- land, USA). EGR1 was cloned into pCMV6-Entry Neomycin Vector. Lipofection of expression vector was performed as recommended in the supplier’s protocol using FuGENE 6 (Roche, Basel, Switzerland). All transfected cells were treated with neomycin constitutively to obtain stable transfectants. EGR stable transfectants were used for invasion assay, examinations of uPA and uPAR expressions, and in vivo experiments. Colony formation assay Colony formation assay was performed as previously described [14]. Briefly, cells were suspended in DMEM containing 0.33% agar and 10% fetal bovine serum and plated onto the bottom layer containing 0.5% agar. The cells were plated at a density of 56103 per well in a 24-well plate, and colonies were counted 14 days later. Each condition was analyzed in triplicate, and all experiments were repeated three times. Invasion assay Invasion of osteosarcoma cells was measured using the BD BioCoatTM BD MatrigelTM Invasion Chamber (BD Bioscience, NJ, U.S.A.) according to the manufacturer’s protocol. Briefly, the cells were transfected with plasmids and selected by neomycin. Osteosarcoma cells were seeded onto the membrane of the upper chamber of the transwell at a concentration of 3–56105/ml in 2 ml of DMEM medium. The medium in the upper chamber was serum-free. The medium in the lower chamber contained 5% fetal calf serum as a source of chemoattractants. Cells that passed through the Matrigel-coated membrane were stained with Diff- Quik (Sysmex, Kobe, Japan) and photographed. Western blot Western blot analysis was performed as previously reported [15]. Briefly, cells were lysed using NP40 lysis buffer (0.5% NP40, 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 3 mM pAPMSF (Wako Chemicals, Kanagawa, Japan), 5 mg/ml aprotinin (Sigma, StLouis, USA), 2 mM sodium orthovanadate (Wako Chemicals, Kanagawa, Japan), and 5 mM EDTA). Lysates were subjected to SDS-PAGE and subsequent immunoblotting was performed. Following antibodies were used: ant- EGR1 and anti-beta actin (Santa Cruz, CA. U.S.A). Detection was performed using the ECL detection system (Amersham, Giles, UK). uPA activity assay uPA activity assay was performed with cell extracts according to the manufacturer’s instructions, with absorption measured at 340 nm (Innovative Research, MI, U.S.A.). The assay measures only the active species of uPA, and a standard curve was generated using recombinant active uPA. The assay conditions were optimized so that the amount of tissue extract or cell extract added gave rise to uPA activity within the linear range of detection. Each reaction was performed in triplicate, and all experiments were repeated for three times. Xenograft model of osteosarcoma For subcutaneous xenograft models, 143B cells were suspended in 100 mL Matrigel (BD, NJ USA.). Cell suspensions were PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16234