Semaphorin 4D Promotes Skeletal Metastasis in Breast Cancer
Fig 2
Sema4D stimulates production of IL-8 from osteoblasts, which promotes osteoclast differentiation.
A. sSema4D treatment of the human osteoblast line HOB induces production of IL-8 in a dose-dependent manner (IL-8 concentration in ng/ml, Y-axis). B. An IL-8 ELISA performed on media conditioned by sSema4D treated HOB confirms this increase (Y-axis, arbitrary units (AU)) out to 24 hrs. C. IL-8 promotes osteoclastogenesis of RAW264.7 cells, particularly in the presence of RANK-L. TRAP assay is shown on top (inset) with the results quantified in the bar graph (average number of TRAP positive osteoclasts/ field, Y-axis). D. RAW264.7 cells are observed to fuse into multinucleated osteoclasts in the presence of IL-8. RANK-L enhances this response. E. Media conditioned by sSema4D treated osteoblasts induces osteoclastogenesis of RAW264.7 cells, a response lost in the presence of IL-8 blocking antibody, demonstrating the importance of this cytokine in differentiation. C, RAW264.7 treated with media conditioned by control osteoblasts (vehicle control), first column. Isotype matched IgG, second column. Anti-IL8 antibody, third and fourth columns. TRAP assay, left; Quantification, right (*, p ≤ 0.05; **, p ≤ 0.01).