Chemotherapy Prevents OS Invasion by EGR1 Figure 4. EGR1 prevents osteosarcoma cell invasion in vitro. Western blot analysis revealed that lysates of EGR1 expression vector-transfected cells were positive for anti-EGR1 antibody (A). Cell invasion assay showed that forced expression of EGR1 decreased 143B, Saos-2, and HOS cell invasion (B) (P,0.05). These experiments were performed in triplicate with similar results [error bars represent mean (SD)]. doi:10.1371/journal.pone.0016234.g004 number of colony formation (Figure S3B). These findings suggest that up-regulation of EGR1 following anti-tumor agent treatment had no effect on osteosarcoma cell growth. Over-expression of EGR1 prevents osteosarcoma cell invasion in vitro To examine the effects of EGR1 up-regulation after anti- tumour agent treatment in modulating the invasive activity of osteosarcoma cells, in vitro invasive activity assays were performed to assess the proportion of osteosarcoma cells transfected with EGR1 expression vector or control vector that invaded through matrigel-coated membranes. Significantly lower proportions of 143B, Saos-2, and HOS cells transiently transfected with EGR1 expression vector migrated through matrigel-coated chambers than osteosarcoma cells transfected with control vector (Figure 4B, C). PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e16234