Fig 2: EVL deficiency impairs VEGFR2 signaling Western blots and quantification of VEGFR2 phosphorylation levels relative to total VEGFR2 levels in WT and EVL-/- endothelial cells under basal and VEGF-stimulated conditions (80 ng/ml); n = 5 independent cell batches, error bars represent SEM; *P < 0.05, one-way ANOVA with Bonferroni’s multi comparison test.Western blots and quantification of ERK1/2 phosphorylation levels relative to total ERK levels in WT and EVL-/- endothelial cells under basal and VEGF-stimulated conditions (80 ng/ml); n = 6 independent cell batches, error bars represent SEM; *P < 0.05, one-way ANOVA with Bonferroni’s multi comparison test.ERK1/2 phosphorylation in P5 retinas of wild-type (WT) and global EVL-/- mice. Retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and antibodies directed against phospho-ERK1/2 (red). Representative images from three independent experiments are shown. Scale bars, 100 µm.Analysis of endothelial ERK1/2 phosphorylation normalized to wild-type littermates. Error bars represent SEM; ***P < 0.001, unpaired Student’s t-test; two different litters.

Fig 3: Antitumor activity of EE02 in an H460 xenograft model. a H460 cells (5 × 106 per flank) were injected into the right flank of mice. When the volume of the tumors reached 50 mm3, the mice were randomly divided into four groups (6 mice in each group). Then, DMSO (negative control), low-dose EE02 (5 mg/kg b.w.), high-dose EE02 (10 mg/kg b.w.) or erlotinib (positive control, 10 mg/kg b.w.) was intraperitoneally injected into the mice every other day. b The volume of each tumor was measured every other day for 20 days. c The body weights of the mice are shown. d The volume of each tumor was measured after tumor extraction at the experimental endpoint. e The tumors excised from the mice after 20 days of treatment are shown. f The expression of Akt, p-Akt, Erk, p-Erk and ki67 in the tumor tissue at the end of the experiments were assessed using IHC staining

Fig 4: VEGF-D induced migration of HMVEC-dLy cells is Erk dependent:(A) Cells were serum starved for 24 h before stimulation with r-VEGF-D (20 ng/ml) for varying lengths of time (0, 15, 30 and 60 minutes) or pre-treated with varying concentrations of Erk inhibitor U0126 (0, 5, 10 and 15 µM) for one hour and further treated with 20 ng/ml r-VEGF-D for 30 minutes. Cell lysates were prepared and analyzed by western blot for Erk1/2 activity. VEGF-D stimulated Erk activation in HMVEC-dLy cells and this stimulation was prevented by pre-treatment with U126. (B) Cell migration was quantified by transwell migration assays; VEGF-D stimulated migration was inhibited with U126 (5–15 µM). 2% FBS served as a positive control. Migration data were represented as a mean ± S.E. for three independent experiments (n = 3). For recombinant VEGF-D stimulation, data were normalised to SFM and for Erk inhibition data were normalised to r-VEGF-D stimulation.

Fig 5: Effect of an ERK-specific inhibitor (SCH 772984) on MMP/TIMP and collagen levels in hypoxic HDFs. Effect of ERK inhibitor treatment on (A) MMP-2, (B) MMP-9, (C) TIMP-1, and (D) type 1 collagen levels in HDFs cultured with (+) or without (-) under 72 hour-hypoxia exposure or SCH 772984 treatment. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.