Figure 1.
CCL5 and CCR5 interaction increased migration of human osteosarcoma cells.
(A&B) MG63 or U20S cells were incubated with various concentrations of CCL5, and in vitro migration and invasion activity measured with the Transwell after 24 h. (C) MG63 cells were incubated with CCL5 (3 ng/ml) for 24 h, and the mRNA expression of CCR1, CCR3 and CCR5 was examined by qPCR. MG63 or U20S cells were pretreated for 30 min with CCR5 Ab, Met-RANTES (D) or transfected with CCR5 siRNA (E) for 24 h. Then they were followed by stimulation with CCL5, and in vitro migration was measured with the Transwell after 24 h. Results of five independent experiments performed are expressed. Results are expressed as the mean ± S.E. *, p<0.05 compared with control; #, p<0.05 compared with CCL5-treated group.
Figure 2.
CCL5 and CCR5 interaction induced migration of human osteosarcoma cells involves up-regulation of αvβ3 integrin.
(A) MG63 or U20S cells were incubated with CCL5 (3 ng/ml) for 24 h, and the mRNA levels of αv, α2, α5, β1, β3 or β5 integrin was determined using qPCR. (B) MG63 or U20S cells were incubated with CCL5 (0.3–3 ng/ml) for 24 h, and the cell surface expression of αvβ3 integrin was determined using flow cytometry. (C) MG63 or U20S cells were pretreated with αvβ3 monoclonal antibody (10 µg/ml), cyclic RGD (100 nM), or cyclic RAD (100 nM) for 30 min followed by stimulation with CCL5. The in vitro migration activity measured after 24 h. (D&E) MG63 or U20S cells were pretreated for 30 min with CCR5 Ab or Met-RANTES followed by stimulation with CCL5. The integrin expression was examined by qPCR and flow cytometry analysis. Results of five independent experiments performed are expressed. Results are expressed as the mean ± S.E. *, p<0.05 compared with control; #, p<0.05 compared with CCL5-treated group.
Figure 3.
MEK is involved in CCL5-mediated migration in human osteosarcoma cells.
(A) MG63 cells were incubated with CCL5 for indicated time intervals, and p-MEK expression was determined by Western blotting. (B–D) MG63 or U20S cells were pretreated with PD98059 and U0126 for 30 min or transfected with dominant negative (DN) mutant of MEK1 for 24 h followed by stimulation with CCL5. The in vitro migration and integrin expression was examined by Transwell, qPCR, and flow cytometry analysis. Results of five independent experiments performed are expressed. Results are expressed as the mean ± S.E. *, p<0.05 compared with control; #, p<0.05 compared with CCL5-treated group.
Figure 4.
ERK is involved in CCL5-mediated migration in human osteosarcoma cells.
(A) MG63 cells were incubated with CCL5 for indicated time intervals, and p-ERK expression was determined by Western blotting. (B) MG63 cells were incubated with CCL5 for indicated time intervals, and ERK kinase activity was examined by ERK kinase assay kit. (C–E) MG63 or U20S cells were transfected with dominant negative (DN) mutant of ERK2 for 24 h followed by stimulation with CCL5. The in vitro migration and integrin expression was examined by Transwell, qPCR, and flow cytometry analysis. Results of five independent experiments performed are expressed. Results are expressed as the mean ± S.E. *, p<0.05 compared with control; #, p<0.05 compared with CCL5-treated group.
Figure 5.
CCL5 induces cell migration and αvβ3 integrin expression through NF-κB.
(A–C) MG63 or U20S cells were pretreated for 30 min with PDTC or TPCK followed by stimulation with CCL5. The in vitro migration and integrin expression was examined by Transwell, qPCR, and flow cytometry analysis. (D) MG63 cells were incubated with CCL5 for indicated time intervals, and p-p65 expression was determined by Western blotting. (E&F) MG63 cells were pretreated with CCR5 Ab, Met-RANTES, PD98059, and U0126 for 30 min or co-transfected with MEK1 and ERK2 mutant or CCR5 siRNA before exposure to CCL5. The NF-κB driven luciferase activity was measured, and the results were normalized to the β-galactosidase activity and expressed as the mean ± S.E. for three independent experiments performed in triplicate. Results of five independent experiments performed are expressed. Results are expressed as the mean ± S.E *, p<0.05 compared with control; #, p<0.05 compared with CCL5-treated group.
Figure 6.
Knockdown of CCL5 inhibited the migratory ability in osteosarcoma cells.
(A) The in vitro migration activity of MG63/control-shRNA and MG63/CCL5-shRNA cells was measured with the Transwell. (B–D) The mRNA levels of CCL5, αv and β3 integrin in MG63/control-shRNA and MG63/CCL5-shRNA cells was examined by qPCR. Results are expressed as the mean ± S.E.