ng2 dsred bac  (Jackson Laboratory)

Journal: International Journal of Molecular Medicine

Article Title: Endothelial UNC5B regulates blood-retinal barrier homeostasis

doi: 10.3892/ijmm.2026.5771

Figure Lengend Snippet: UNC5B specifically regulates endothelium-pericyte interactions with no significant effect on pericyte function. HRMECs were transfected with lentiviral shUNC5B or shC and puromycin was used to select stably transfected HRMECs. (A) Following transduction, HRMECs were co-cultured with HRMVPCs for 6 h and then stained with NG2 (HRMVPCs) and IB4 (HRMECs) to detect the recruitment of pericytes toward endothelial cells. Scale bar, 100 μ m. (B) For BRB model formation, HRMVPCs were seeded in the lower compartment of the Transwell insert 1 h prior to the addition of HRMECs. Co-cultures were maintained for 2 days in complete DMEM. (C) Barrier properties of the BRB model were assessed by permeability analysis. Subsequently, HRMVPCs were transfected with lentiviral shUNC5B or shC, and puromycin was used to select stably transfected HRMVPCs. UNC5B expression was examined by (D) reverse transcription-quantitative PCR and (E) western blotting. (F) Apoptosis (measured by PI incorporation), (G) cell proliferation (measured by EdU incorporation) and (H) migration were examined using the described assays, and the results were quantified. Scale bar, 100 μ m. Data are presented as the mean ± SD. n=5 per group, except for western blot experiments (n=4). All statistical comparisons in this figure were performed between the C group and each of the other groups using one-way ANOVA followed by Dunnett's multiple comparisons test. * P<0.05 vs. C. BRB, blood-retinal barrier; C, control; ECs, endothelial cells; EdU, 5-ethynyl-2'-deoxyuridine; HRMEC, human retinal microvascular endothelial cell; HRMVPC, human retinal microvascular pericyte; IB4, isolectin B4; NG2, neuron-glial antigen 2; ns, not significant; PCs, pericytes; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; UNC5B, unc-5 netrin receptor B.

Article Snippet: Subsequently, the endothelial cells were fluorescently labeled with isolectin B4 (IB4; 1:50; cat. no. I21411 ; Thermo Fisher Scientific, Inc.), whereas pericytes were stained with an anti-neuron-glial antigen 2 (NG2) antibody (1:200; cat. no. sc-53389; Santa Cruz Biotechnology, Inc.) at 4°C overnight.

Techniques: Transfection, Stable Transfection, Transduction, Cell Culture, Staining, Permeability, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Migration, Control, shRNA

Journal: International Journal of Molecular Medicine

Article Title: Endothelial UNC5B regulates blood-retinal barrier homeostasis

doi: 10.3892/ijmm.2026.5771

Figure Lengend Snippet: UNC5B silencing in endothelial cells disrupts blood-retinal barrier homeostasis in DR model mice and abrogates the protective effect of high-concentration netrin-1. At week 4 after successful DR model induction, different concentrations of netrin-1 (5, 50, 500, 1,000 and 5,000 ng/ml) were injected intravitreally. Retinal tissues were collected after ≥12 weeks of DR model induction for subsequent analysis. (A) EB staining was used to observe retinal vascular leakage in the different groups. Scale bar, 500 μ m. At week 4 after DR model induction, control shRNA-AAV carrying an endothelial cell-specific promoter sequence (DR + shC), 1,000 ng/ml netrin-1 (DR + Netrin-1), UNC5B shRNA-AAV carrying an endothelial cell-specific promoter sequence (DR + shUNC5B) or a combination of 1,000 ng/ml netrin-1 and UNC5B shRNA-AAV (DR + shUNC5B + Netrin-1) were injected. After ≥12 weeks of DR model induction, retinal tissues were collected for analysis. (B) EB staining showed retinal vascular leakage in the different groups. Scale bar, 500 μ m. (C) Periodic acid-Schiff staining revealed the formation of acellular capillaries (arrows) and the number of pericytes ('P') in the retinal tissues. Scale bar, 50 or 20 μ m. (D) Whole-mount retinal immunofluorescence staining showed the pericyte coverage in the retinas of the different groups (red, NG2; green, IB4). Scale bar, 200 μ m. The data are presented as the mean ± SD. n=5 per group. All statistical comparisons in this figure were performed among all groups using one-way ANOVA followed by Tukey's multiple comparisons test. * P<0.05 vs. WT. # P<0.05 vs. DR/DR + shC. AAV, adeno-associated virus; DR, diabetic retinopathy; EB, Evans Blue; IB4, isolectin B4; NG2, neuron-glial antigen 2; ns, not significant; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; UNC5B, unc-5 netrin receptor B; WT, wild-type.

Article Snippet: Subsequently, the endothelial cells were fluorescently labeled with isolectin B4 (IB4; 1:50; cat. no. I21411 ; Thermo Fisher Scientific, Inc.), whereas pericytes were stained with an anti-neuron-glial antigen 2 (NG2) antibody (1:200; cat. no. sc-53389; Santa Cruz Biotechnology, Inc.) at 4°C overnight.

Techniques: Concentration Assay, Injection, Staining, Control, shRNA, Sequencing, Immunofluorescence, Virus

Journal: International Journal of Molecular Medicine

Article Title: Endothelial UNC5B regulates blood-retinal barrier homeostasis

doi: 10.3892/ijmm.2026.5771

Figure Lengend Snippet: UNC5B overexpression in endothelial cells maintains blood-retinal barrier homeostasis in DR model mice. At week 4 after successful DR model induction, DR mice received a single retro-orbital injection of adeno-associated virus carrying an endothelial cell-specific promoter. Mice were assigned to two groups: One with overexpression of UNC5B (DR + oeUNC5B) and the other receiving an empty vector as a control (DR + NC). Retinal tissues were collected after ≥12 weeks of DR model induction for subsequent analysis. (A) EB staining showed retinal vascular leakage in the different groups. Scale bar, 500 μ m. (B) Periodic acid-Schiff staining revealed acellular capillary formation (arrows) and pericyte numbers ('P') in the retinas of the different groups. Scale bar, 50 or 20 μ m. (C) Whole-mount retinal immunofluorescence staining showed pericyte coverage in the retinas of the different groups (red, NG2; green, IB4). Scale bar, 200 μ m. The data are presented as the mean ± SD. n=5 per group. All statistical comparisons in this figure were performed among all groups using one-way ANOVA followed by Tukey's multiple comparisons test. * P<0.05 vs. WT. # P<0.05 vs. DR + NC. DR, diabetic retinopathy; EB, Evans Blue; IB4, isolectin B4; NC, negative control; NG2, neuron-glial antigen 2; oe, overexpression; UNC5B, unc-5 netrin receptor B; WT, wild-type.

Article Snippet: Subsequently, the endothelial cells were fluorescently labeled with isolectin B4 (IB4; 1:50; cat. no. I21411 ; Thermo Fisher Scientific, Inc.), whereas pericytes were stained with an anti-neuron-glial antigen 2 (NG2) antibody (1:200; cat. no. sc-53389; Santa Cruz Biotechnology, Inc.) at 4°C overnight.

Techniques: Over Expression, Injection, Virus, Plasmid Preparation, Control, Staining, Immunofluorescence, Negative Control