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. 2015 Oct 26;291(5):2087–2106. doi: 10.1074/jbc.M115.696419

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© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — Melanoma contacts increased endothelial gap formation and permeability in a B-Raf(V600E)- and PAR-1-dependent manner. A, gaps in AJ were formed at the sites of melanoma adhesion in the presence of PFP. 1 × 10⁶ Lu1205 or UACC903 cells that were stained with DiI were co-cultured with a HUVEC monolayer for 60 min in the presence (+PFP) or absence of PFP (−PFP) before the VE-cadherin was stained. With PFP, Lu1205 or UACC903 contacts induced gap formation and reduced VE-cadherin staining at the cell periphery. White arrows indicate the locations of junction dissociation and tumor pseudopod protrusion. Bar, 5 μm. B, percent endothelial gaps significantly increased upon melanoma-HUVEC co-culture in the presence of plasma. 1 × 10⁶ WM35, A375M, UACC903, and Lu1205 melanoma cells were co-cultured with a HUVEC monolayer for 60 min in the presence or absence of PFP before the endothelial gap area was determined. *, p < 0.05 compared with −PFP; †, p < 0.05 compared with no TC control. C, HUVECs were grown on transwell inserts with 0.4-μm pores before being left alone or co-cultured with WM35 or Lu1205 melanoma cells with or without PFP for the indicated time periods. HUVEC monolayer TER was measured as described under “Experimental Procedures.” The time-dependent change of the electrical resistance of HUVEC monolayer stimulated with 80 ng/ml VEGF served as a positive control. D, silencing B-Raf(V600E) with siRNA in late-stage melanoma cells, Lu1205 and UACC903, resulted in a dramatic decrease in gap formation in the presence of, but not in the absence of, PFP. *, p < 0.05 compared with control cases. si^V600EBRAF, B-Raf(V600E) siRNA. E, depletion of TF with siRNA or blocking of TF with antibody-attenuated Lu1205/PFP-induced gap formation. *, p < 0.05 compared with control cases (buffer, scrambled, or isotype control). F, anti-coagulant, hirudin, reduced Lu1205/PFP-induced gap formation. *, p < 0.05 compared with vehicle control. G, HUVECs were grown on transwell inserts with 0.4-μm pores before co-culturing with Lu1205 melanoma cells transfected with buffer, scrambled siRNA, B-Raf(V600E) siRNA (si^V600EBRAF), or siTF, or treated with vehicle control or 40 units/ml hirudin with PFP for indicated time periods. HUVEC monolayer TER was measured as described in “Experimental Procedures.” The time-dependent changes in the electrical resistance of the HUVEC monolayer, which was left alone or stimulated with 80 ng/ml VEGF, served as controls. H, depletion of PAR-1 with siRNA attenuated Lu1205- or UACC903/PFP-induced gap formation. *, p < 0.05 compared with control cases (buffer or scrambled). Knockdown efficiency was assessed by PCR. siPAR-1, PAR-1 siRNA. I, HUVECs transfected with buffer, scrambled siRNA, or siPAR-1 were grown on transwell inserts with 0.4-μm pores. Then they were co-cultured with Lu1205 melanoma cells for indicated time periods. HUVEC monolayer TER was measured as described under “Experimental Procedures.” The time-dependent change in the electrical resistance of the HUVEC monolayer, which was stimulated with 80 ng/ml VEGF, served as a control. Values are mean ± S.E. from at least three independent experiments.

FIGURE 2. — Melanoma contacts increased endothelial gap formation and permeability in a B-Raf(V600E)- and PAR-1-dependent manner. A, gaps in AJ were formed at the sites of melanoma adhesion in the presence of PFP. 1 × 10⁶ Lu1205 or UACC903 cells that were stained with DiI were co-cultured with a HUVEC monolayer for 60 min in the presence (+PFP) or absence of PFP (−PFP) before the VE-cadherin was stained. With PFP, Lu1205 or UACC903 contacts induced gap formation and reduced VE-cadherin staining at the cell periphery. White arrows indicate the locations of junction dissociation and tumor pseudopod protrusion. Bar, 5 μm. B, percent endothelial gaps significantly increased upon melanoma-HUVEC co-culture in the presence of plasma. 1 × 10⁶ WM35, A375M, UACC903, and Lu1205 melanoma cells were co-cultured with a HUVEC monolayer for 60 min in the presence or absence of PFP before the endothelial gap area was determined. *, p < 0.05 compared with −PFP; †, p < 0.05 compared with no TC control. C, HUVECs were grown on transwell inserts with 0.4-μm pores before being left alone or co-cultured with WM35 or Lu1205 melanoma cells with or without PFP for the indicated time periods. HUVEC monolayer TER was measured as described under “Experimental Procedures.” The time-dependent change of the electrical resistance of HUVEC monolayer stimulated with 80 ng/ml VEGF served as a positive control. D, silencing B-Raf(V600E) with siRNA in late-stage melanoma cells, Lu1205 and UACC903, resulted in a dramatic decrease in gap formation in the presence of, but not in the absence of, PFP. *, p < 0.05 compared with control cases. si^V600EBRAF, B-Raf(V600E) siRNA. E, depletion of TF with siRNA or blocking of TF with antibody-attenuated Lu1205/PFP-induced gap formation. *, p < 0.05 compared with control cases (buffer, scrambled, or isotype control). F, anti-coagulant, hirudin, reduced Lu1205/PFP-induced gap formation. *, p < 0.05 compared with vehicle control. G, HUVECs were grown on transwell inserts with 0.4-μm pores before co-culturing with Lu1205 melanoma cells transfected with buffer, scrambled siRNA, B-Raf(V600E) siRNA (si^V600EBRAF), or siTF, or treated with vehicle control or 40 units/ml hirudin with PFP for indicated time periods. HUVEC monolayer TER was measured as described in “Experimental Procedures.” The time-dependent changes in the electrical resistance of the HUVEC monolayer, which was left alone or stimulated with 80 ng/ml VEGF, served as controls. H, depletion of PAR-1 with siRNA attenuated Lu1205- or UACC903/PFP-induced gap formation. *, p < 0.05 compared with control cases (buffer or scrambled). Knockdown efficiency was assessed by PCR. siPAR-1, PAR-1 siRNA. I, HUVECs transfected with buffer, scrambled siRNA, or siPAR-1 were grown on transwell inserts with 0.4-μm pores. Then they were co-cultured with Lu1205 melanoma cells for indicated time periods. HUVEC monolayer TER was measured as described under “Experimental Procedures.” The time-dependent change in the electrical resistance of the HUVEC monolayer, which was stimulated with 80 ng/ml VEGF, served as a control. Values are mean ± S.E. from at least three independent experiments.