Figure 5 Netrin-4 induces in vitro... | American Society of Hematology

Figure 5

$Figure 5. Netrin-4 induces in vitro lymphatic permeability. (A) Induction of GTP-RhoA and Rac1 by Netrin-4, VEGF-A, and VEGF-C treatment of HMVEC-dLys. (B) Stimulation of the phosphorylation of Tyrosine 416 of Src kinase family (SFK, Tyr416) and the Tyrosin 861 but not the Tyrosine 391 of focal adhesion kinase (FAK; Tyr861 and Tyr391) by Netrin-4 (500 ng/mL) and VEGF-C (500 ng/mL). (C) Measurement of the electrical resistance of the cell monolayer over 24 hours using the ECIS system or (D) immunostained using an anti–VE-cadherin antibody to visualize cell junctions (scale bar: 50 μm) of HMVEC-dLys seeded either on Fibronectin or Fibronectin plus Netrin-4. (E) Membrane fraction proteins prepared from HMVEC-dLys seeded as previously mentioned in panels C and D analyzed for ZO-1, VE-cadherin, and beta-catenin expression (equivalent loading assessed by coomassie blue staining). (F) Control, Netrin-4, VEGF-C overexpressing MCF7 tumor sections stained for the cell junction protein ZO-1 or the lymphatic marker LYVE1 (scale bar: 20 μm). Data presented in panels A through E are from 1 experiment and representative of 2 independent experiments. Pictures were taken on an Olympus IX71 microscope, at 400× magnification using a DP30BW Olympus camera and the MicroSuite Basic Edition Olympus software.$

Netrin-4 induces in vitro lymphatic permeability. (A) Induction of GTP-RhoA and Rac1 by Netrin-4, VEGF-A, and VEGF-C treatment of HMVEC-dLys. (B) Stimulation of the phosphorylation of Tyrosine 416 of Src kinase family (SFK, Tyr416) and the Tyrosin 861 but not the Tyrosine 391 of focal adhesion kinase (FAK; Tyr861 and Tyr391) by Netrin-4 (500 ng/mL) and VEGF-C (500 ng/mL). (C) Measurement of the electrical resistance of the cell monolayer over 24 hours using the ECIS system or (D) immunostained using an anti–VE-cadherin antibody to visualize cell junctions (scale bar: 50 μm) of HMVEC-dLys seeded either on Fibronectin or Fibronectin plus Netrin-4. (E) Membrane fraction proteins prepared from HMVEC-dLys seeded as previously mentioned in panels C and D analyzed for ZO-1, VE-cadherin, and beta-catenin expression (equivalent loading assessed by coomassie blue staining). (F) Control, Netrin-4, VEGF-C overexpressing MCF7 tumor sections stained for the cell junction protein ZO-1 or the lymphatic marker LYVE1 (scale bar: 20 μm). Data presented in panels A through E are from 1 experiment and representative of 2 independent experiments. Pictures were taken on an Olympus IX71 microscope, at 400× magnification using a DP30BW Olympus camera and the MicroSuite Basic Edition Olympus software.

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