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UNC5B maintains the normal barrier function of endothelial cells. (A) Fluorescence microscopy image of HRMECs transfected with green fluorescence-labeled lentivirus-coated UNC5B shRNA. Scale bar, 100 μ m. HRMECs were transfected with lentiviral shUNC5B or shC, and puromycin was used to select stably transfected HRMECs. UNC5B (B) mRNA and (C) protein expression was examined by reverse transcription-quantitative PCR and western blotting. (D) Live and apoptotic cells were assessed using <t>PI/Calcein-AM</t> staining (green, live cells; red, dead or dying cells). Scale bar, 100 μ m. (E) mRNA and (F) protein expression levels of barrier regulators were examined. (G) HRMECs were incubated with RBITC-BSA for 6 h. The internalized RBITC-BSA was imaged by microscopy and the intensity of the internalized RBITC-BSA was quantitated using ImageJ (red, RBITC-BSA; blue, DAPI). Scale bar, 20 μ m. (H) Schematic illustration of the experimental procedure for assessing HRMEC monolayer permeability and the corresponding quantitative results. 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. C, control; GFP, green fluorescent protein; HRMEC, human retinal microvascular endothelial cell; ns, not significant; OD, optical density; PLVAP, plasmalemma vesicle-associated protein; RBITC, rhodamine B isothiocyanate; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; UNC5B, unc-5 netrin receptor B; ZO-1, zonula occludens 1.
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UNC5B maintains the normal barrier function of endothelial cells. (A) Fluorescence microscopy image of HRMECs transfected with green fluorescence-labeled lentivirus-coated UNC5B shRNA. Scale bar, 100 μ m. HRMECs were transfected with lentiviral shUNC5B or shC, and puromycin was used to select stably transfected HRMECs. UNC5B (B) mRNA and (C) protein expression was examined by reverse transcription-quantitative PCR and western blotting. (D) Live and apoptotic cells were assessed using <t>PI/Calcein-AM</t> staining (green, live cells; red, dead or dying cells). Scale bar, 100 μ m. (E) mRNA and (F) protein expression levels of barrier regulators were examined. (G) HRMECs were incubated with RBITC-BSA for 6 h. The internalized RBITC-BSA was imaged by microscopy and the intensity of the internalized RBITC-BSA was quantitated using ImageJ (red, RBITC-BSA; blue, DAPI). Scale bar, 20 μ m. (H) Schematic illustration of the experimental procedure for assessing HRMEC monolayer permeability and the corresponding quantitative results. 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. C, control; GFP, green fluorescent protein; HRMEC, human retinal microvascular endothelial cell; ns, not significant; OD, optical density; PLVAP, plasmalemma vesicle-associated protein; RBITC, rhodamine B isothiocyanate; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; UNC5B, unc-5 netrin receptor B; ZO-1, zonula occludens 1.
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UNC5B maintains the normal barrier function of endothelial cells. (A) Fluorescence microscopy image of HRMECs transfected with green fluorescence-labeled lentivirus-coated UNC5B shRNA. Scale bar, 100 μ m. HRMECs were transfected with lentiviral shUNC5B or shC, and puromycin was used to select stably transfected HRMECs. UNC5B (B) mRNA and (C) protein expression was examined by reverse transcription-quantitative PCR and western blotting. (D) Live and apoptotic cells were assessed using <t>PI/Calcein-AM</t> staining (green, live cells; red, dead or dying cells). Scale bar, 100 μ m. (E) mRNA and (F) protein expression levels of barrier regulators were examined. (G) HRMECs were incubated with RBITC-BSA for 6 h. The internalized RBITC-BSA was imaged by microscopy and the intensity of the internalized RBITC-BSA was quantitated using ImageJ (red, RBITC-BSA; blue, DAPI). Scale bar, 20 μ m. (H) Schematic illustration of the experimental procedure for assessing HRMEC monolayer permeability and the corresponding quantitative results. 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. C, control; GFP, green fluorescent protein; HRMEC, human retinal microvascular endothelial cell; ns, not significant; OD, optical density; PLVAP, plasmalemma vesicle-associated protein; RBITC, rhodamine B isothiocyanate; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; UNC5B, unc-5 netrin receptor B; ZO-1, zonula occludens 1.
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UNC5B maintains the normal barrier function of endothelial cells. (A) Fluorescence microscopy image of HRMECs transfected with green fluorescence-labeled lentivirus-coated UNC5B shRNA. Scale bar, 100 μ m. HRMECs were transfected with lentiviral shUNC5B or shC, and puromycin was used to select stably transfected HRMECs. UNC5B (B) mRNA and (C) protein expression was examined by reverse transcription-quantitative PCR and western blotting. (D) Live and apoptotic cells were assessed using <t>PI/Calcein-AM</t> staining (green, live cells; red, dead or dying cells). Scale bar, 100 μ m. (E) mRNA and (F) protein expression levels of barrier regulators were examined. (G) HRMECs were incubated with RBITC-BSA for 6 h. The internalized RBITC-BSA was imaged by microscopy and the intensity of the internalized RBITC-BSA was quantitated using ImageJ (red, RBITC-BSA; blue, DAPI). Scale bar, 20 μ m. (H) Schematic illustration of the experimental procedure for assessing HRMEC monolayer permeability and the corresponding quantitative results. 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. C, control; GFP, green fluorescent protein; HRMEC, human retinal microvascular endothelial cell; ns, not significant; OD, optical density; PLVAP, plasmalemma vesicle-associated protein; RBITC, rhodamine B isothiocyanate; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; UNC5B, unc-5 netrin receptor B; ZO-1, zonula occludens 1.
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UNC5B maintains the normal barrier function of endothelial cells. (A) Fluorescence microscopy image of HRMECs transfected with green fluorescence-labeled lentivirus-coated UNC5B shRNA. Scale bar, 100 μ m. HRMECs were transfected with lentiviral shUNC5B or shC, and puromycin was used to select stably transfected HRMECs. UNC5B (B) mRNA and (C) protein expression was examined by reverse transcription-quantitative PCR and western blotting. (D) Live and apoptotic cells were assessed using <t>PI/Calcein-AM</t> staining (green, live cells; red, dead or dying cells). Scale bar, 100 μ m. (E) mRNA and (F) protein expression levels of barrier regulators were examined. (G) HRMECs were incubated with RBITC-BSA for 6 h. The internalized RBITC-BSA was imaged by microscopy and the intensity of the internalized RBITC-BSA was quantitated using ImageJ (red, RBITC-BSA; blue, DAPI). Scale bar, 20 μ m. (H) Schematic illustration of the experimental procedure for assessing HRMEC monolayer permeability and the corresponding quantitative results. 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. C, control; GFP, green fluorescent protein; HRMEC, human retinal microvascular endothelial cell; ns, not significant; OD, optical density; PLVAP, plasmalemma vesicle-associated protein; RBITC, rhodamine B isothiocyanate; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; UNC5B, unc-5 netrin receptor B; ZO-1, zonula occludens 1.
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UNC5B maintains the normal barrier function of endothelial cells. (A) Fluorescence microscopy image of HRMECs transfected with green fluorescence-labeled lentivirus-coated UNC5B shRNA. Scale bar, 100 μ m. HRMECs were transfected with lentiviral shUNC5B or shC, and puromycin was used to select stably transfected HRMECs. UNC5B (B) mRNA and (C) protein expression was examined by reverse transcription-quantitative PCR and western blotting. (D) Live and apoptotic cells were assessed using PI/Calcein-AM staining (green, live cells; red, dead or dying cells). Scale bar, 100 μ m. (E) mRNA and (F) protein expression levels of barrier regulators were examined. (G) HRMECs were incubated with RBITC-BSA for 6 h. The internalized RBITC-BSA was imaged by microscopy and the intensity of the internalized RBITC-BSA was quantitated using ImageJ (red, RBITC-BSA; blue, DAPI). Scale bar, 20 μ m. (H) Schematic illustration of the experimental procedure for assessing HRMEC monolayer permeability and the corresponding quantitative results. 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. C, control; GFP, green fluorescent protein; HRMEC, human retinal microvascular endothelial cell; ns, not significant; OD, optical density; PLVAP, plasmalemma vesicle-associated protein; RBITC, rhodamine B isothiocyanate; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; UNC5B, unc-5 netrin receptor B; ZO-1, zonula occludens 1.

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 maintains the normal barrier function of endothelial cells. (A) Fluorescence microscopy image of HRMECs transfected with green fluorescence-labeled lentivirus-coated UNC5B shRNA. Scale bar, 100 μ m. HRMECs were transfected with lentiviral shUNC5B or shC, and puromycin was used to select stably transfected HRMECs. UNC5B (B) mRNA and (C) protein expression was examined by reverse transcription-quantitative PCR and western blotting. (D) Live and apoptotic cells were assessed using PI/Calcein-AM staining (green, live cells; red, dead or dying cells). Scale bar, 100 μ m. (E) mRNA and (F) protein expression levels of barrier regulators were examined. (G) HRMECs were incubated with RBITC-BSA for 6 h. The internalized RBITC-BSA was imaged by microscopy and the intensity of the internalized RBITC-BSA was quantitated using ImageJ (red, RBITC-BSA; blue, DAPI). Scale bar, 20 μ m. (H) Schematic illustration of the experimental procedure for assessing HRMEC monolayer permeability and the corresponding quantitative results. 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. C, control; GFP, green fluorescent protein; HRMEC, human retinal microvascular endothelial cell; ns, not significant; OD, optical density; PLVAP, plasmalemma vesicle-associated protein; RBITC, rhodamine B isothiocyanate; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; UNC5B, unc-5 netrin receptor B; ZO-1, zonula occludens 1.

Article Snippet: The cells were stained with a 1:1 mixture of PI and calcein-AM dye (cat. no. C1371; Beyotime Biotechnology) at 37°C for 10 min. Fluorescence microscopy (Olympus Corporation) was then employed to visualize the staining patterns, with viable cells emitting green fluorescence under a 490-nm excitation filter and necrotic cells emitting red fluorescence under a 545-nm excitation filter.

Techniques: Fluorescence, Microscopy, Transfection, Labeling, shRNA, Stable Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Staining, Incubation, Permeability, Control