Journal: International Journal of Molecular Sciences

Article Title: Hypoxia Inhibits Cell Cycle Progression and Cell Proliferation in Brain Microvascular Endothelial Cells via the miR-212-3p/MCM2 Axis

doi: 10.3390/ijms24032788

Figure Lengend Snippet: Functional analysis of differentially expressed genes (DEGs) in bEnd.3 cells under 24 h hypoxia. ( A ) Volcano map of differentially expressed genes between the control and hypoxia conditions. ( B ) Heatmap of the top upregulated and downregulated genes. ( C ) Gene ontology (GO) term analysis of differentially expressed genes. ( D ) Top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. ( E ) Top 10 upregulated pathways from gene set enrichment analysis (GSEA). ( F ) Top 10 downregulated pathways from GSEA.

Article Snippet: The mouse brain microvascular endothelial bEnd.3 cell line was obtained from iCell (Shanghai, China).

Techniques: Functional Assay

Journal: bioRxiv

Article Title: On the shuttling across the blood-brain barrier via tubules formation: mechanism and cargo avidity bias

doi: 10.1101/2020.04.04.025866

Figure Lengend Snippet: Schematic of the in vitro model of BBB used to assess transcytosis (a) . Bar chart showing the apparent permeability coefficient ( P ) of 4 and 70 kDa dextrans across bEnd3. Data represented as mean ± SD ( n = 9) (b) . Confocal images of polarised bEnd3 showing expression of PECAM-1, claudin-5 and ZO-1. PECAM-1, claudin-5 and ZO-1 are shown in green and cell nuclei stained with DAPI (blue). Scale: 20 nm (c) .

Article Snippet: Mouse brain endothelial cells bEnd3 were used between passage 20-30. bEnd3 were grown in DMEM supplemented with 10% (v/v) FBS and 100 IUL mL −1 penicillin/100 mg mL −1 streptomycin.

Techniques: In Vitro, Permeability, Expressing, Staining

Journal: bioRxiv

Article Title: On the shuttling across the blood-brain barrier via tubules formation: mechanism and cargo avidity bias

doi: 10.1101/2020.04.04.025866

Figure Lengend Snippet: Confocal micrograph of polarized bEnd3 showing colocalisation of Cy5-labelled A 22 -P (in grey) and LRP1 (in yellow) after 60 minutes of incubation (a) . Cell nuclei were counterstained with DAPI (in blue). Higher magnification of the co-localised Cy5-labelled A 22 -P and LRP1 on bEnd3 displaying the colocalisation values, r (a1) . Competition assay of angiopep-2 and A 22 -P. Comparison of intracellular fluorescence intensity of angiopep-2 or A 22 -P when incubated alone or co-incubated with polarised bEnd3. Data represented as mean ± SD ( n = 3). * P <0.05 and ** P <0.01 (b) . Confocal image of clathrin (in green), Cy5-labelled A 22 -P (in red) and actin (in cyan) in bEnd3 treated with Cy5-labelled A 22 -P (c) . Higher manification of clathrin (in green) and Cy5-labelled A 22 -P (in red) staining on bEnd3 showing a partial colocalisation (c1-c4) . Confocal images of polarised bEnd3 incubated with Cy5-labelled A 22 -P (in red) for 10 and 60 minutes and stained for caveolin-1 (in green). Cell nuclei is stained with DAPI (blue). Colocalisation values r are displayed on the top corner of each image (d) . 3D rendering of bEnd3 incubated with Cy5-labelled A 22 -P (in red) and stained for caveolin-1 (green) (e) . Confocal micrographs of polarised bEnd3 stained for F-actin (Phalloidin in green) and incubated with Cy5-labelled A 22 -P (in red) for 10, 30 and 60 minutes. Nuclei stained with DAPI (blue). Colocalisation values r for F-actin and Cy5-labelled A 22 -P shown at the bottom of each merged image (f) . A higher magnification image showing colocalisation of Cy5-labelled A 22 -P and F-actin (f1) .

Article Snippet: Mouse brain endothelial cells bEnd3 were used between passage 20-30. bEnd3 were grown in DMEM supplemented with 10% (v/v) FBS and 100 IUL mL −1 penicillin/100 mg mL −1 streptomycin.

Techniques: Incubation, Competitive Binding Assay, Comparison, Fluorescence, Staining

Journal: bioRxiv

Article Title: On the shuttling across the blood-brain barrier via tubules formation: mechanism and cargo avidity bias

doi: 10.1101/2020.04.04.025866

Figure Lengend Snippet: Quantification of free cholesterol released into the media after depletion from the plasma membrane by incubation with methyl- β -cyclodextrin (CD) in the apical or basal side of the transwell. Data is represented as mean ± SD ( n = 3) (a) . Fold-change in the level of cholesterol in the apical and basal side of the transwell membrane after incubation with CD. Data is represented as mean ± SD ( n = 3). * P <0.05, ** P <0.01, **** P <0.0001 (b) . Confocal images of Cy5-labelled A 22 -P (red) in cholesterol-depleted bEnd3 cells either treated with CD in the apical (+CD A) or basal (+CD BL) side of transwell. Caveolin-1 is represented in green. Scale bar: 5 μ m (c) .

Article Snippet: Mouse brain endothelial cells bEnd3 were used between passage 20-30. bEnd3 were grown in DMEM supplemented with 10% (v/v) FBS and 100 IUL mL −1 penicillin/100 mg mL −1 streptomycin.

Techniques: Clinical Proteomics, Membrane, Incubation

Journal: bioRxiv

Article Title: On the shuttling across the blood-brain barrier via tubules formation: mechanism and cargo avidity bias

doi: 10.1101/2020.04.04.025866

Figure Lengend Snippet: Expression of syndapin-2 on shRNA-transfected bEnd3 cells quantified by western blot. Levels of syndapin-2 on bEnd3 either treated with shRNA control or syndapin-2 were normalised to wild-type cells. Data is represented as mean ± SD ( n = 6) (a) . Bar charts showing apparent permeability of 4 and 70 kDa dextrans across bEnd3 transfected with shRNA control or shRNA for syndapin-2 knockdown. Data represented as mean ± SD ( n = 6) (b) . Apparent permeability of Cy7-labelled A 22 -P across control and syndapin-2 knockout bEnd3 cells. Data represented as mean ± SD ( n = 6). *** P <0.001 (c) .

Article Snippet: Mouse brain endothelial cells bEnd3 were used between passage 20-30. bEnd3 were grown in DMEM supplemented with 10% (v/v) FBS and 100 IUL mL −1 penicillin/100 mg mL −1 streptomycin.

Techniques: Expressing, shRNA, Transfection, Western Blot, Control, Permeability, Knockdown, Knock-Out

Journal: bioRxiv

Article Title: On the shuttling across the blood-brain barrier via tubules formation: mechanism and cargo avidity bias

doi: 10.1101/2020.04.04.025866

Figure Lengend Snippet: Confocal images and corresponding 3D rendering of polarised bEnd3 incubated with Cy5-labelled A 22 -P (red) for 10 minutes, 1, 3 and 6 hours. Tight junction, claudin-5, is represented in green and the nuclei counterstained with DAPI (in blue). Scale bar: 25 μ m (a) . Real-time 4D confocal imaging of transcytosis of Cy5-labelled A 22 -P. Heat map of Cy5-labelled A 22 -P fluorescence in the extended transwell area over time obtained through 4D imaging (b) .

Article Snippet: Mouse brain endothelial cells bEnd3 were used between passage 20-30. bEnd3 were grown in DMEM supplemented with 10% (v/v) FBS and 100 IUL mL −1 penicillin/100 mg mL −1 streptomycin.

Techniques: Incubation, Imaging, Fluorescence