subcutaneously inoculated in nude mice. Three weeks after inoculation, 4 mg/kg doxorubicin was administered by intraper- itoneal injection. One day after treatment, mice were scarified and tumors were examined. For metastasis experiments, 143B cells (56105) were transfected with GFP lentiviral particles (Santa Cruz, CA. U.S.A). Stably-GFP-expressing 143B cells (16106) were mixed with a collagen gel in a 1:1 volume, and inoculated into the left knee joint of 6-weeks-old nude mice. Five weeks after inoculation, the mice were sacrificed. GFP-positive-143B cells were counted in 50 ml blood aspirates from hearts using the M165 FC microscope (Leica Microsystems, Wild Heerbrugg, Switzer- land). Metastatic nodules in the lungs were evaluated by direct microscopic visualization using an M165 FC microscope. Lung metastasis area was calculated by Lumina Vision (Mitani Corporation, Tokyo, Japan). All experimental procedures were performed in compliance with the guiding principles for the Care and Use of Animals described in the American Journal of Physiology and with the Guidelines established by the Institute of Laboratory Animal Sciences, Faculty of Medicine, Kagoshima University (approval number: 20064). All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize possible alternatives to in vivo techniques. ELISA Expression levels of uPA and uPAR proteins were assayed using specific enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions (Abnova, Taipei, Taiwan). Cell lysates were collected by EGR1 stable transfected osteosarcoma cells. Statistics Each sample was analyzed in triplicate, and experiments were repeated three times. In all figures, error bars are standard deviations. All statistical analyses were performed using Microsoft Office Excel (Microsoft, Albuquerque, New Mexico, USA) and STASTISCA (StatSoft, Tulsa, OK, USA). Differences between mean values were evaluated by the unpaired t-test, and differences in frequencies by Fisher’s exact test. Differences were considered significant at P,0.05. Results EGR1 is down-regulated in osteosarcoma cell lines and patient’ specimens Real-time PCR was performed to examine the gene expression of EGR1 in osteoblast and osteosarcoma cell lines including NHOst, 143B, Saos-2, HOS, MG63, and NOS-1. Real-time PCR revealed that the 5 of 5 osteosarcoma cell lines exhibited 0.002- to 0.369-fold decreased in expression of EGR1 (Figure 1A). In addition, we performed real-time PCR using patient’ biopsy specimens. Real-time PCR revealed that EGR1 was decreased 0.01-to 0.2-fold in 8 of 10 human biopsy specimens (Figure 1B). These findings suggest that the EGR1 is down-regulated in human osteosarcomas. Anti-tumour agent treatment promoted up-regulation of EGR1 To examine the effects of anti-tumour agents on EGR1 expression, we performed real-time PCR after anti-tumour agent treatment. We attempt to clarify the changes in EGR1 expression following low-dose anti-tumor agent treatment, and determined anti-tumor drug concentrations required to prevent osteosarcoma cell proliferation. MTT assay revealed that 250 ng/ml cisplatin, 1 ng/ml methotrexate, 50 ng/ml etoposide, or 10 ng/ml doxo- Chemotherapy Prevents OS Invasion by EGR1 Figure 1. Down-regulation of EGR1 in human osteosarcoma. Total RNA extracted from osteosarcoma cell lines (A) and osteosarcoma patients’ biopsy specimens (B) were analyzed by real-time PCR. Results revealed that 5 of 5 human osteosarcoma cell lines and 8 of 10 human biopsy specimens of osteosarcoma had decreased EGR1 expression. The comparative Ct (DDCt) method was used to determine fold change in expression using GAPDH. These experiments were performed in triplicate with similar results. doi:10.1371/journal.pone.0016234.g001 rubicin treatment did not prevent 143B cell growth. Growth of Saos-2 cells was not inhibited by 1 ng/ml methotrexate, 5 ng/ml methotrexate, 10 ng/ml methotrexate, 50 ng/ml etoposide, or 10 ng/ml doxorubicin. On the other hand, Growth of 143B cell and Saos-2 cell was inhibited by higher dose of each drug (Figure 2 A, B). Following 24 h treatment with these concentrations of anti-tumor drugs, EGR1 was up-regulated (Figure 3 A–D). Following 48 h or 5 days treatment, cisplatin, methotrexate, etoposide or doxorubicin increased EGR1 expres- sion in 143B cell and Saos-2 cells (Figure S1). We next examined the expression of EGR1 following chemotherapy in biopsy specimens. Specimens of OS6, OS8, and OS9 tumors were obtained during tumor resection in osteosarcoma patients who received chemotherapy. We compared the EGR1 expressions in the biopsy specimens and the resected tumor specimens obtained from these patients. In 3 of 3 patient’ specimens examined, EGR1 expression was increased 7.87- to 1.71 following chemotherapy (Figure S2A). To examine the expression of EGR1 following low- dose chemotherapy in vivo, we used a novel osteosarcoma murine xenograft model with 143B cells. We injected 4 mg/kg doxoru- bicin which is less than one-hundred dose of COSS-86 protocol for osteosarcoma patients. Real-time PCR showed that low dose doxorubicin treatment promoted up-regulation of EGR1 in vivo (Figure S2B). PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e16234