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Fig. 5 | Journal of Hematology & Oncology

Fig. 5

From: Tie2-mediated vascular remodeling by ferritin-based protein C nanoparticles confers antitumor and anti-metastatic activities

Fig. 5

Endothelial PAR-3 is required for TFMG-induced normalization of vasculature. a Schematic diagram of the endothelial cell/cancer cell (EC) co-culture system. EA.hy926 cells (5 × 105) were co-cultured with LLC cells (2.5 × 105) in Transwell plates (pore size, 3 μm). b Permeability assay of TFG and TFMG using EC co-cultures. EC co-cultures were treated with TFG (100 nM) or TFMG (100 nM) for 48 h, and the permeability (%) of FITC-dextran was measured relative to untreated control co-cultures. B, Blank (no EA.hy926, no LLC); E, endothelial cell only (EA.hy926 seeded onto the Transwell membrane, no LLC); *P < 0.0001 vs. B; #P < 0.0001 vs. E; $P < 0.0001 vs. control (n = 3). c, d Permeability assay of TFMG using EC co-cultures to demonstrate the time- and dose-dependent effects. EC co-cultures were treated with 100 nM TFMG for 12, 24, or 48 h (c) and with 10, 100, and 1000 nM TFMG for 24 h (d), and the permeability (%) of FITC-dextran was measured relative to untreated control co-cultures (n = 3). e Effect of siRNA-mediated endothelial PAR-1, PAR-2, and PAR-3 silencing on TFMG-induced reduction of in vitro permeability. EC co-cultures, with or without siRNA-mediated silencing of endothelial PAR-1, PAR-2, or PAR-3, were treated with 100 nM TFMG for 24 h, and the permeability (%) of FITC-dextran was measured relative to untreated control co-cultures. *P < 0.0001 vs. untreated control EC co-culture; #P < 0.001 vs. TFMG-treated EC co-culture; $P < 0.001 vs. TFMG-treated EC co-culture with siNC (negative control siRNA) silencing; ns, no significant difference vs. TFMG-treated EC-cultures with or without siNC silencing (n = 5). f, g Co-immunoprecipitation assay of PAR-1 and PAR-3 following TFG or TFMG treatment. EA.hy926 cells were treated with TFG or TFMG (100 nM) for 24 h. The PAR-1/PAR-3 interaction was investigated by immunoprecipitation with anti-PAR3 antibody followed by western blot analysis using anti-PAR-1 antibody in TFG/TFMG-treated or untreated control cells (f). Relative binding of PAR-1/PAR-3 after TFG or TFMG treatment was quantitated using GelQuant software (g). Data information: Data are presented as the mean ± SD. Significant enrichment: *P < 0.05; ***P < 0.001; ****P < 0.0001 (c, d, f, g) (Sidak’s multiple comparisons test)

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