Fig 2: Distribution of M1 and M2 macrophages with Dll4 expression in laser-induced CNV observed under an immunofluorescence microscope. One eye of each C57BL/6 mouse was exposed to a laser to induce CNV, and the other eye was left untreated. Eyecups were collected on day 7 after laser coagulation. (A and B; thin white arrows) Dll4-positive M1 macrophages were marked by double staining for CD80 (green) and Dll4 (red). (C and D; thick white arrows) Dll4-positive M1 macrophages in cryosections of the CNV lesion were found expressed at low levels either untreated or after laser coagulation. Dll4-positive M2 macrophages were marked by double staining for CD206 (green) and Dll4 (red). (E) Dll4-positive M2 macrophage number in cryosections of the CNV lesion was significantly increased after laser coagulation compared with untreated eyes. Each experiment was repeated three times. Data are expressed as the means ± SD (n=3 mice per group). **P<0.01 as indicated. Scale bar, 50 µm. CNV, choroidal neovascularization; Dll4, delta-like 4; ns, no significance.

Fig 3: MMD-Dll4 pathway expression in RPE-choroid tissues of laser-induced CNV. RPE-choroid tissues were resected from mice exhibiting laser-induced CNV. (A) The expression of MMD, Dll1 and Dll4 was determined by western blotting, with ß-actin as a control. Each experiment was repeated three times. Statistical analysis was performed using ImageJ software. (B) Reverse transcription-quantitative PCR was performed, and the expression of MMD, Dll4 and Dll1 was normalized to ß-actin. Data are expressed as the mean ± SD (n=3 mice per group). *P<0.05 and **P<0.01 as indicated. Dll1, delta-like 1; Dll4, delta-like 4; IL-4, adenovirus-expressed IL-4 vector; LacZ and LacZ, adenovirus-expressed ß-galactosidase; MMD, monocyte to macrophage differentiation-associated; ns, no significance; RPE, retinal pigment epithelium; WT, wild-type.

Fig 4: Loss of EHD2 blunts Dll4 endocytosis. (A) Western blot of EHD2 after siRNA (si) treatment in comparison to scramble (Scram) control in Human vein endothelial cells. (B) Schematic of pHrodo-labeled delta-like ligand 4 (Dll4) antibody. PHrodo gains fluorescent intensity with increasing endosomal pH, thus used as a metric of endocytosis. (C) Relative internalization of pHrodo-Dll4 pulse-chase over time between indicated siRNA groups. IgG was used as a non-specific internalization control. A minimum of 50 cells were used per group. Order of comparisons (top to bottom): Scram si vs. EHD2 si; Scram IgG vs. pHrodo-Dll4 with EHD2 si. (D) Relative internalization of pHrodo-Dll4 pulse-chase over time between indicated groups. A minimum of 50 cells were used per group. Order of comparisons (top to bottom): DMSO vs. Dynosore; DMSO vs. MßCD. (E) Schematic of recombinant Notch intracellular domain (NECD) functionalized to microbead and labeled with pHrodo. (F) Relative internalization of functionalized NECD pulse-chase over time between indicated siRNA groups. A minimum of 50 cells were used per group. Comparisons: Scram si vs. EHD2 si treated cells. (G) Relative internalization of functionalized NECD pulse-chase over time between indicated siRNA groups. A minimum of 50 cells were used per group. Order of comparisons (top to bottom): Scram si vs. Dll4 si; Scram si vs. Jagged1 (Jag1) si; Scram si vs. Dll4 and Jag1 si treated cells. (H) Relative internalization of functionalized NECD pulse-chase over time between indicated siRNA groups. A minimum of 50 cells were used per group. Order of comparisons (top to bottom): Scram si vs. caveolin1 (Cav1) si; Scram si vs. clathrin light chain B (CLTB) si; Scram si vs. AP2 Associated Kinase 1 (AAK1) si treated cells. (I) Antibody feeding assay comparing internalized to surface-bound ratio Dll4 between indicated groups. N, number of cells. (J) Western blot of Dll4 levels across indicated treatment groups. (K) Relative expression of Hes1 compared to GAPDH control in indicated si treatment groups. N, number of replicates. *p < .05, **p < .01, ***p < .001, ****p < .0001. ns, non-significant. Error bars are SEM. All experiments were done at minimum in triplicate

Fig 5: Membranous Dll4 localizes with EHD2 and caveolin-1. (A) Representative image of proximity ligation assay (PLA). Cells were stained as indicated and PLA reaction is marked by red puncta. Control condition was not transfected. Dll4-myc condition was only transfected with Dll4-myc and stained for endogenous EHD2. (B) Graph of number of PLA puncta by condition. Control condition was not transfected. EHBP1 condition was transfected with EHBP1-HA and Dll4-myc. Dll4-myc condition was only transfected with Dll4-myc and stained for endogenous EHD2. (C) Schematic of engineered Dll4 (top). Bottom, cartoon of pH-dependent function of GFP variant pHluorin tag. PHluorin fluoresces on the membrane at neutral pH but is quenched when internalized into acidic endosomes allowing for visualization of only membranous Dll4. (D) Representative image of endothelial cell (HUVEC) stained for caveolin-1 (Cav1) expressing pHluorin-Dll4 (pH-Dll4) and RFP-EHD2. (E) Representative image of cell stained for actin expressing pH-Dll4 and RFP-EHD2. (F) Representative image of cell stained for clathrin expressing pH-Dll4 and RFP-EHD2. (G) Proportion of coincidence of clathrin (y-axis) and EHD2 (x-axis) around Dll4 puncta. (H) Pearson's correlation between indicated proteins. N, number of cells. Boxes denote magnified images on right. Yellow arrowheads show areas of pH-Dll4 puncta. ***p < .001. Error bars are 95% confidence intervals. All experiments were done at minimum in triplicate