Journal: Antioxidants

Article Title: Endothelial Notch Signaling Regulates the Function of the Retinal Pigment Epithelial Barrier via EC Angiocrine Signaling

doi: 10.3390/antiox12111979

Figure Lengend Snippet: Primer sequences used in this study.

Article Snippet: Then, the concentration of secreted HBEGF in the media was measured using the human HBEGF ELISA kit (Boster, Wuhan, China).

Techniques: Sequencing

Journal: Antioxidants

Article Title: Endothelial Notch Signaling Regulates the Function of the Retinal Pigment Epithelial Barrier via EC Angiocrine Signaling

doi: 10.3390/antiox12111979

Figure Lengend Snippet: Endothelial Notch signaling affects RPE barrier function by regulating the HBEGF paracrine of EC. ( A ) Analysis for previously published transcriptome sequencing data of normal HUVECs and DAPT treated HUVECs: the GSEA results for ECM ( left ) and heatmap of the expression of ECM-related genes ( right ). ( B ) mRNA and protein levels of HBEGF in normal HUVECs and DAPT-treated HUVECs. ( C ) mRNA and protein levels of HBEGF in normal HUVECs and NICD adenovirus-infected HUVECs. ( D ) The secretion levels of HBEGF protein in normal HUVECs and DAPT-treated HUVECs or NICD adenovirus-infected HUVECs were tested using an ELISA kit. ( E ) Evaluating the RPE barrier function in RPE cells cultured in normal medium, RPE cultured in the EC-conditioned medium, RPE cultured in Notch signaling-inhibited EC-conditioned medium, and RPE cultured in Notch signaling-inhibited EC-conditioned medium supplemented with recombinant HBEGF (r-HBEGF) using ZO-1 staining (top), fibronectin staining ( middle ), and collagen IV staining (bottom). The number of RPEs with intact tight junctions were quantitatively compared. Fibronectin- and collagen IV-positive areas were quantitatively compared. Scale bars: 50 µm. ( F ) Evaluating the RPE barrier function in the RPE, RPE with HUVECs, RPE with siHBEGF-treated HUVECs, and RPE with siNC-treated HUVECs using ZO-1 staining (top), fibronectin staining ( middle ), and collagen IV staining (bottom). The number of RPEs with intact tight junctions were quantitatively compared. Fibronectin- and collagen IV-positive areas were quantitatively compared. Scale bars: 50 µm. Three biological replicates were performed and values are presented as the mean ± SEM ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then, the concentration of secreted HBEGF in the media was measured using the human HBEGF ELISA kit (Boster, Wuhan, China).

Techniques: Sequencing, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant, Staining

Journal: Antioxidants

Article Title: Endothelial Notch Signaling Regulates the Function of the Retinal Pigment Epithelial Barrier via EC Angiocrine Signaling

doi: 10.3390/antiox12111979

Figure Lengend Snippet: r-HBEGF improved the RPE barrier dysfunction of Notch signaling deletion mice. ( A ) In the SI-induced RPE barrier disfunction model, whole-mount choroidal staining for EC-specific Notch signaling deletion mice CDH5-CRE-RBPJ f/f intravitreally injected with IgG or r-HBEGF, and control mice (RBPJ f/+ ) at p30, were analyzed using ZO-1 staining. The number of RPE cells with intact tight junctions were quantitatively compared. Red circles indicate the location of the optic disk. Scale bars: 50 µm. ( B ) In the laser-induced CNV model, whole-mount choroidal staining for EC-specific Notch signaling deletion mice CDH5-CRE-RBPJ f/f intravitreally injected with IgG or r-HBEGF and control mice (RBPJ f/+ ) were analyzed using IB-4 staining. IB4 positive areas were quantitatively compared. Three biological replicates were performed and values are presented as the mean ± SEM ( n = 3). Scale bars: 150 µm. * p < 0.05, ** p < 0.01.

Article Snippet: Then, the concentration of secreted HBEGF in the media was measured using the human HBEGF ELISA kit (Boster, Wuhan, China).

Techniques: Staining, Injection, Control

Journal: Antioxidants

Article Title: Endothelial Notch Signaling Regulates the Function of the Retinal Pigment Epithelial Barrier via EC Angiocrine Signaling

doi: 10.3390/antiox12111979

Figure Lengend Snippet: EC regulates MMP-9 expression in RPE cells via Endothelial Notch signaling-driven HBEGF secretion. ( A ) Protein interaction prediction analysis results of HBEGF and MMP-9, using the String database. ( B ) mRNA level, protein level, and enzyme activity level of MMP-9 in the RPE, RPE with HUVEC, and RPE with Notch signaling-inhibited HUVECs were tested via qRT-PCR, Western blot, and gelatin assay kit. ( C ) mRNA levels of MMP-9 in normal HUVECs and DAPT-treated HUVECs were tested via qRT-PCR. ( D , E ) mRNA and protein levels of MMP-9 in the RPE, RPE with HUVECs, RPE with siNC-treated HUVECs, and RPE with siHBEGF-treated HUVECs were tested via qRT-PCR and Western blot. ( F ) Protein levels of MMP-9 in RPE cells cultured in normal medium, RPE cultured in EC-conditioned medium, RPE cultured in Notch signaling-inhibited EC-conditioned medium, and RPE cultured in Notch signaling-inhibited EC-conditioned medium supplemented with recombinant HBEGF (r-HBEGF) were tested using Western blot. Three biological replicates were performed and values are presented as the mean ± SEM ( n = 3). NS, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then, the concentration of secreted HBEGF in the media was measured using the human HBEGF ELISA kit (Boster, Wuhan, China).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Western Blot, Cell Culture, Recombinant

Journal: eLife

Article Title: Thalamocortical axons control the cytoarchitecture of neocortical layers by area-specific supply of VGF

doi: 10.7554/eLife.67549

Figure Lengend Snippet:

Article Snippet: For electroporation-based cell ablation, pCAG- DTR was constructed as follows: a cDNA fragment containing the coding region of human HBEGF (GenBank accession number: BC033097) was obtained from a commercial supplier (clone ID 100067676, DNAFORM).

Techniques: CRISPR, Recombinant, Plasmid Preparation, Expressing, Sequencing, Imaging, Software, Staining