Chemotherapy Prevents OS Invasion by EGR1 Figure 2. Osteosarcoma cell growth following anti-tumor drug treatment. MTT assay showed that growth at 48 h of 143B cells was not inhibited by 250 ng cisplatin, 1 ng/ml methotrexate, 50 ng/ml etoposide, or 10 ng/ml doxorubicin. Growth of 143B cell was inhibited by higher dose of each drug (A) (P,0.05). Growth at 48 h 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. Growth of Saos-2 cell was inhibited by higher dose of each drug (B) (P,0.05). The experiment was performed in triplicate with similar results [error bars represent mean (SD)]. doi:10.1371/journal.pone.0016234.g002 Over-expression of EGR1 does not prevent osteosarcoma growth It has been reported that EGR1 over-expression suppresses the growth of cell in soft agar and tumor growth in nude mice [10,16]. We therefore, transfected the EGR1 expression vector and examined osteosarcoma cell growth. Western blot analysis showed up-regulation of EGR1 in 143B, Saos-2, and HOS cells (Figure 4A). MTT assay revealed that forced expression of EGR1 did not prevent osteosarcoma growth in vitro (Figure S3A). We next examined the effects of EGR1 on anchorage- independent osteosarcoma growth. Colony formation assay revealed that forced expression of EGR1 did not affect the PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16234