Chemotherapy Prevents OS Invasion by EGR1 Down-regulation of uPA and uPAR by EGR1 We then examined the cellular mechanisms by which EGR1 exerts its effects on osteosarcoma cell invasion. Several investiga- tions have shown that EGR1 plays an important role in the control of tumor metastasis through regulation of cancer invasion-related genes, including TGF-b1, thrombospondin-1, and plasminogen activator inhibitor-1 [17,18]. We examined whether EGR1 affects the expression of cancer invasion-related genes. Real-time PCR revealed that forced expression of EGR1 in 143B, Saos-2, and HOS osteosarcoma cell lines decreased the expression of uPA and uPAR (Figure 5). ELISA revealed that forced expression of EGR1 decreased the expression of uPA and uPAR proteins (Figure S4). Further, we examine the effects of anti-tumour agents on uPA and uPAR expression in vitro, we performed real-time PCR after anti- tumor agent treatment. Treatment of low dose anti-tumor drugs decreased the expression of uPA and uPAR (Figure S5). To examine the effects of low dose chemotherapy on uPA and uPAR expression in vivo, we used osteosarcoma murine xenograft model with 143B cells. Nude mice were treated with 4 mg/kg doxorubicin. Real-time PCR showed that low dose doxrubicin Figure 5. EGR1 decreased expression of uPA and uPAR. We examined whether EGR1 affects the expression of uPA and uPAR. RNA was prepared from control vector or EGR1 expression vector stably transfected cells. Real-time PCR revealed that forced expression of EGR1 decreased uPA and uPAR expression in 143B, Saos-2, and HOS cells (P,0.05). The comparative Ct (DDCt) method was used to determine fold change in expression using GAPDH. These experiments were performed in triplicate with similar results [error bars represent mean (SD)]. doi:10.1371/journal.pone.0016234.g005 PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16234