Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Monocyte Chemoattractant Protein-1 promotes cancer cell migration via c-Raf/MAPK/AP-1 pathway and MMP-9 production in osteosarcoma

doi: 10.1186/s13046-020-01756-y

Figure Lengend Snippet: MCP-1-induced osteosarcoma migration required the CCR2 receptor. a CCR2 and CCR4 inhibitors (400 nM) were added to the cells. After MCP-1 stimulation, MG63 migration was measured. b and c MMP-9 mRNA and protein expression were detected, respectively, with CCR2 and CCR4 inhibitor treatment and MCP-1 stimulation. (D and E) After the MG63 cells were treated with CCR2 mAb for 30 min, cell migration and MMP-9 mRNA expression were measured, respectively. f - h The MG63 cells were transfected with CCR2 siRNA for 24 h, and the CCR2 protein expression, cell migration and MMP-9 mRNA expression were measured with or without MCP-1 stimulation. i CCR2 protein production was detected in the osteoblast cell line hFOB 1.19 and the osteosarcoma cell lines MG63, U2OS and HOS were assessed using Western blotting. j CCR2 protein production was detected in the MG63 cells with different migratory abilities (M10, M20, and M30) through Western blotting. Results are expressed as mean ± SEM, n = 4. * p < 0.05 compared with control, IgG, control siRNA, hFOB1.19 or MG63 groups, respectively; # p < 0.05 compared with the MCP-1-treated group

Article Snippet: All siRNAs which target specific genes (si-c-Jun: sc-29,223; si-MMP9: sc-29,400; si-CCR2: sc-270,220; si-control: sc-44,231) were purchased from Santa Cruz Biotechnology. p-c-Raf (Ser338) was purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Migration, Expressing, Transfection, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Monocyte Chemoattractant Protein-1 promotes cancer cell migration via c-Raf/MAPK/AP-1 pathway and MMP-9 production in osteosarcoma

doi: 10.1186/s13046-020-01756-y

Figure Lengend Snippet: 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

Article Snippet: All siRNAs which target specific genes (si-c-Jun: sc-29,223; si-MMP9: sc-29,400; si-CCR2: sc-270,220; si-control: sc-44,231) were purchased from Santa Cruz Biotechnology. p-c-Raf (Ser338) was purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Migration, Expressing, Western Blot, Transfection, Incubation, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Immunoprecipitation