Figure 6.

Exosomes secreted from OS cells regulate fibroblasts activation to foster lung metastasis. A. Examination of COL6A1 protein in the culture medium (CM) derived from COL6A1-overexpressing cells (CM/COL6A1) and control cells (CM/NC) by western blotting. B. Cell migration potential was determined in U2OS and Saos-2 cells incubated with CM/COL6A1 and CM/NC according to transwell assays. C. Confocal imaging showed the delivery of PKH67-labeled exosomes (green) to MRC5 cell (Scale bar, 100 µm). D. COL6A1 expression of MRC5 treated with exosomes released by indicated cells were detected by western blot. E. Migration ability of MRC5 treated with equal quantities of exosomes derived from indicated OS cells, detected by transwell assay and wound healing assay. F. MRC5 treated with exosomes released by indicated OS cells were assessed for their ability to contract collagen. Collagen contraction was quantified by the Image J software. G. Indicated gene expression of MRC5 treated with exosomes released by COL6A1-transfection OS cells or control were detected by qRT-PCR analysis. H. ELISA assay was performed to detect the expression of IL-6, IL-8 and TGF-β in MRC5 cells treated by indicated cells. I. NF-kB expression and transcription activation was detected by western blot and dual luciferase reporter assay in MRC5 cells treated by indicated cells. J. The rate of lung metastasis after tail-vein injection of indicated cells (n = 6 each group). Representative photographs of H&E staining (Scale bars: 400 µm) and COL6A1, a-SMA immunohistochemistry staining (Scale bars: 100 µm) in lung metastases tissues from mice orthotopically inoculated with indicated treated cells. Data represent the mean ± SD of 3 separate determinations. *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t test.