Figure 7

VEGF in the microenvironment elicits OPN nuclear accumulation. (a) Representative semi-qualitative assay of cytokines in homogenates from indicated lungs using mouse angiogenesis antibody array. Schematic diagram of the array is shown and spots were scanned for densitometry analysis using the Quantity One software (Bio-Rad, Hercules, CA, USA). Data represent mean±S.D. (b) Immunofluorescence staining of OPN and GM130 in HCCLM3 cells after treatment with Leptin (100 ng/ml), FGFa (50 ng/ml), or VEGF (200 ng/ml). Scale bar, 10 μm. (c) Representative images of IHC staining in indicated metastases. Scale bars, 100 μm. (d) Representative bioluminescent images and the log2 luminescent photon flux of mice, which were treated as the diagram. *P<0.05. (e) Immunoblot analysis of phosphoserine and OPN in HCCLM3 cells stimulated as indicated. The ratio of gray values of phosphoserine and OPN were compared. Ctr, control. (f, g and h) Immunofluorescence staining of OPN and GM130 in HCCLM3 cells. (f) Cells were treated with PKC inhibitors, Tamoxifen (6 μ M), Sotrasaurin (5 μM), or Myreciten (10 μM). (g) Cells were treated with the indicated antibody (300 ng/ml) and VEGF (200 ng/ml). (h) Cells were treated with the PLCγ inhibitor U73122 (20 μM) and VEGF (200 ng/ml). Scale bars, 10 μm. (i) The schematic model of sOPN and iOPN as facilitators of tumor metastasis by inducing EMT and MET separately during the invasion-colonization cascade