Fig. 7.

Knockdown of MCP-1 inhibited the migratory ability of osteosarcoma cells. a-b MCP-1-overexpressing MG-63 cells were established, using pCDNA3.1 vector. Protein and mRNA expression of MCP-1 and migratory activity were examined by western blot, q-PCR, and Transwell assay. c Treating MG63 cells with shRNA against MCP-1 decreased the protein level of MCP-1 as well as that of MMP-9. d shRNA knockdown of MCP-1 decreased the in vitro migration activity of osteosarcoma cells. e To induce pulmonary metastases, MG63 cells were injected into the mouse tail vein and those mice were sacrificed after 28 days later with developed lung metastatic nodules. Compared to the control mice, there are fewer and smaller tumors which were seen on the lungs of mice injected with osteosarcoma cells transfected with shRNA against MCP-1. f H&E staining and IHC of lung metastatic nodules of MG63 injected mice. g The lungs of MG63 injected mice were removed and inflated with 10% paraformaldehyde fixative. The number of lung metastatic nodules was counted under a dissecting microscope. h MCP-1 and MMP-9 expression levels in specimens were determined in a tumor tissue microarray by IHC staining. The stained specimens were photographed using an optical microscope. i and j IHC stain intensities were scored from 1 to 5 to quantify MCP-1 and MMP-9 expression levels in different stages. k Correlation between MCP-1 and MMP-9 expression in human osteosarcoma specimens. Results are shown as mean ± SEM. n = 4. *p < 0.05 compared with pcDNA3.1-empty, control or MG63-control shR groups