Fig. 6.

MCP-1-promoted osteosarcoma migration and MMP-9 upregulation required c-Jun/AP-1 involvement. a A migration assay was performed in the MG63 cells with curcumin (1 μM) and tanshinone IIA (5 μM) pretreated for 30 min with MCP-1 stimulation. b and c The MG63 cells were treated with curcumin and tanshinone IIA for 30 min, and MMP-9 mRNA and protein expression were measured. d At different MCP-1 stimulation durations (0, 10, 15, 30, and 60 min), c-Jun phosphorylated and total proteins in both the cytosol and nucleus were measured using Western blotting. e The MG63 cells were transfected with control and c-Jun siRNA and then incubated for 24 h. A migration assay was performed to measure osteosarcoma migratory ability. f-g The MG63 cells were transfected with control and c-Jun siRNA and incubated for 24 h. Cell migration and MMP-9 mRNA expression was measured with MCP-1 stimulation. h AP-1 luciferase activity was measured in the MG63 cells at various concentrations of MCP-1 stimulation. The results were normalized according to the β-galactosidase activity. i The MG63 cells were treated with MCP-1 stimulation and different inhibitors (CCR2 inhibitor, GW5074, U0126, PD98059, SB203580, and SP600125), and AP-1 luciferase activity was measured and normalized according to the β-galactosidase activity. j The MG63 cells were treated with the CCR2 inhibitor and GW5074 for 30 min, respectively, and c-Jun phosphorylated and total protein production were measured using Western blotting. k and l After the MG63 cells were pretreated with the CCR2 inhibitor, GW5074, U0126, PD98059, SB203580, and SP600125 for 30 min, MCP-1 was added to stimulate the cells for 120 min. The chromatin immunoprecipitation assay was then performed with anti-c-Jun immunoprecipitation. To verify equal loading amount (input), 1% of the precipitated chromatin was applied. Results are expressed as mean ± SEM, n = 4. *p < 0.05 compared with control or control siR groups; #p < 0.05 compared with the MCP-1-treated group