Fig 2: Depletion of VEGFR2 levels blunts retinal neovascularization.a Upper panel: Equal amount of protein from control and the indicated time periods of VEGFA-treated HRMVECs were analyzed for phospho and total VEGFR1, 2 and 3 levels. Lower panel: The cell extracts were analyzed for VEGFR2 and VEGFR3 complex formation. b HRMVECs were transfected with siControl or siVEGFR2 (100 nM), quiesced, treated with and without VEGFA (40 ng/ml) for 10 min (for pPKC?) or 120 min (for JunB and VEGFR3) and cell extracts were prepared and analyzed by western blotting for the indicated proteins. c Retinas from normoxic and 24 h (i.e., at P13) of hypoxic WT mice pups that received siControl or siVEGFR2 (1 µg/0.5 µl/eye) by intravitreal injections at P10 and P11 were isolated, extracts were prepared and analyzed by western blotting for the indicated proteins. d Eyes from normoxic and 72 h (i.e., at P15) of hypoxic WT mice pups that received siControl or siVEGFR2 (1 µg/0.5 µl/eye) by intravitreal injections at P11, P12 and P13 were enucleated, fixed, sections were made and coimmunostained for CD31 and VEGFR2 (left panel) and CD31 and VEGFR3 (right panel). e Eyes from normoxic and 24 h (i.e., at P13) of hypoxic WT mice pups that were injected intravitreally with vehicle or 0.05 µg/0.5 µl/eye of soluble VEGFR2 at P11 and P12 were enucleated, retinas were isolated, protein extracts were prepared and analyzed by western blotting for the indicated proteins using their specific antibodies. f Eyes from normoxic and 24 h of hypoxic mice pups that were injected intravitreally with siControl or siVEGFR2 (0.5 µg/0.5 µl/eye) at P11 and P12 were enucleated, retinas were isolated, protein extracts were prepared and analyzed by western blotting for VEGFR3 levels using its specific antibodies and the blot was normalized for ß-tubulin. g All the conditions were the same as in (d) except that sections were coimmunostained for CD31 and Ki67. The bar graph shows quantification of proliferating ECs per section. h All the conditions were the same as in (d) except that eyes were enucleated at P17, fixed, retinas were isolated, stained with isolectin B4, flat mounts were made and examined for filopodia at 40× magnification (scale bar, 50 µm). Bar graph shows quantification of the number of filopodia/unit vessel length. i All the conditions were the same as in (h) except that the flat mounts were examined for retinal vascularization. Retinal vascularization is shown in the first column at 2.5× magnification (scale bar, 500 µm). Neovascularization is highlighted in red in the second column. The third column shows the selected rectangular areas of the images in the first column at 10× magnification (scale bar, 200 µm). j,k Retinal neovascularization (j) and avascular area (k) were determined as described in “Materials and Methods.” The values are presented as mean ± SD. *p < 0.01 vs normoxia; **p < 0.01 vs siControl + hypoxia.

Fig 3: The putative model of the mechanism by which PTEN regulates the invasiveness of pNETs via DUSP19-mediated VEGFR3 dephosphorylation. PTEN positively regulates DUSP19 expression. DUSP19 binds directly to VEGFR3 to dephosphorylate it. Low expression of PTEN induces downregulation of DUSP19 and suppresses DUSP19-mediated dephosphorylation of VEGFR3, which leads to increased VEGFR3 phosphorylation and activation of its downstream ERK signaling pathway and EMT molecules (vimentin and SLUG) to enhance tumor invasiveness

Fig 4: VEGFR3 phosphorylation is responsible for pNET cell invasiveness in a VEGFC-independent manner. A The relative migration and invasion abilities of QGP-1 cells with (shPTEN) and without (shLuc) knockdown of PTEN and treated with the VEGFR3-Fc chimera protein (shPTEN-R3Fc) or the VEGFR3 inhibitor MAZ51 (shPTEN-MAZ51). Migration: shLuc vs. shPTEN, P = 0.021; shPTEN vs. shPTEN-MAZ51, P = 0.030; Wilcoxon rank-sum test. Invasion: shLuc vs. shPTEN, P = 0.021; shPTEN vs. shPTEN-MAZ51, P = 0.029; Wilcoxon rank-sum test. B The protein level of phosphorylated VEGFR3 in QGP-1 cells with and without knockdown of PTEN and treated with the VEGFR3-Fc chimera protein or the VEGFR3 inhibitor MAZ51. C The relative migration and invasion abilities of QGP-1 cells with (shPTEN) and without (shLuc) knockdown of PTEN and with (shPTEN/VEGFR3) or without knockdown of VEGFR3. Migration: shPTEN vs. shPTEN/VEGFR3, P = 0.030; Wilcoxon rank-sum test. Invasion: shPTEN vs. shPTEN/VEGFR3, P = 0.030; Wilcoxon rank-sum test. The protein level of phosphorylated VEGFR3 in QGP-1 cells with and without knockdown of PTEN and with or without knockdown of VEGFR3 was shown in right panel

Fig 5: DUSP19 dephosphorylates VEGFR3 via a direct receptor tyrosine kinase–protein phosphatase interaction. A Western blot analysis of the VEGFR3 immunoprecipitates showed that DUSP19 was present in the immunocomplex from QGP-1 cells with knockdown of vector control (shLuc) but not present in the immunocomplex from QGP-1 cells with DUSP19 knockdown (left panel). Western blot analysis of the DUSP19 immunoprecipitates showed that VEGFR3 was present in the immunocomplex from QGP-1 cells with knockdown of vector control (shLuc) but not present in the immunocomplex from QGP-1 cells with VEGFR3 knockdown (right panel). Immunoprecipitation with IgG was used as negative control. B VEGFR3 and DUSP19 were shown to be colocalized on the cell membrane of QGP-1 cells by immunofluorescence staining. C Confocal microscope images of XY and XZ sections. D The phospho-signal of the phospho-VEGFR3 peptide was reduced by the addition of recombinant DUSP19 (P = 0.021, Wilcoxon rank-sum test)