Journal: Molecular Oncology
Article Title: Sphingosine‐1‐phosphate suppresses chondrosarcoma metastasis by upregulation of tissue inhibitor of metalloproteinase 3 through suppressing miR‐101 expression
Figure Lengend Snippet: S1P‐induced TIMP ‐3 expression is mediated through the c‐Src/ MAPK signaling axis in human chondrosarcoma cells. (A) Starved JJ 012 cells were incubated with 10 μ m of S1P for 10, 15, 30, 60, and 120 min, and the phosphorylation of c‐Src, MEK , and ERK was examined by immunoblotting using anti‐phospho c‐Src, MEK , or ERK antibodies, followed by reprobing against total c‐Src, MEK , ERK , and β‐actin to show the equal loading amounts. (B) The JJ 012 cells were transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h, and the protein expressions of c‐Src, MEK , and ERK were then determined to show the knockdown efficiency. Starved JJ 012 (open bar) and SW 1353 (closed bar) cells were pretreated with 10 μ m of PP 2, PD 98059, and U0126 for 1 h or transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h, followed by treatment with 10 μ m of S1P for another 24 h, and expressions of miR‐101 (C), TIMP ‐3 (E), and MMP ‐2 (F) mRNA , as well as the protein expressions of TIMP ‐3 and MMP ‐2 (G), and migratory activity (H), were then determined by real‐time PCR , immunoblotting, and transwell analyses, respectively. (D) JJ 012 and SW 1353 cells were transfected with plasmids harboring with WT ‐ TIMP ‐3 3′ UTR , followed by treatment with 10 μ m of PP 2, PD 98059, and U0126 for 1 h or transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h prior to incubation with 10 μ m of S1P for another 24 h. The promoter activity of TIMP ‐3 was then determined by luciferase activity assay. (I) Starved JJ 012 cells were pretreated with 10 μ m of PP 2 and PD 98059 for 1 h, followed by treatment with 10 μ m of S1P for another 10 min, and the phosphorylation of Src, MEK , and ERK was then determined by immunoblotting analysis. Cells without treatment were used as the untreated control (set to 1 or 100), and data were shown as multiples of that. The results are expressed as mean ± SEM . * P
Article Snippet: The anti‐rabbit sphingosine kinase 1, a tissue inhibitor of metalloproteinase 3, was obtained from Genetex (Irvine, CA, USA); anti‐rabbit p‐c‐Src and p‐MEK were obtained from Cell Signaling Technology (Danvers, MA, USA); anti‐rabbit p‐ERK, ERK, MEK, MMP‐2, and c‐Src were obtained from Santa Cruz (Santa Cruz, CA, USA).
Techniques: Expressing, Incubation, Transfection, Activity Assay, Real-time Polymerase Chain Reaction, Luciferase