human brain microvascular endothelial cell line hbec 5i  (ATCC)

Journal: Nature Communications

Article Title: Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2

doi: 10.1038/s41467-025-68058-9

Figure Lengend Snippet: A , Experimental timeline for inflammatory insult with TNF-α and FGF2 co-treatment. Once confluent (4 days), HBEC-5i or bEnd.3 were pretreated with FGF2 or vehicle for one hour and then stimulated with TNF-α or vehicle for up to 7 days. FGF2 alters mouse ( B ) and human ( C ) endothelial transcription in response to acute TNF-α stimulation. FGF2 promotes faster restoration of TNF-α-induced Cldn5 loss in mouse ( D ) and human ( E ) endothelial cells. Chronic stimulation with TNF-α leads to a reduction in endothelial monolayer integrity measured by trans-endothelial electrical resistance (TEER) in mouse ( F ) and human ( G ) endothelial cells. FGF2 co-treatment preserves normal TEER despite TNF-α. H 7 days of TNF-α treatment promotes spikes and discontinuities in Cldn5 tight junction strands in bEnd.3, which is reversed by FGF2 treatment (scalebar = 20 μm). Data represent mean ± s.e.m., and each experiment was replicated at least twice on independent samples. Group comparisons were evaluated with two-way ANOVA followed by Bonferroni’s post hoc tests; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The human brain microvascular endothelial cell line HBEC-5i (ATCC CRL-3245, male donor according to https://www.cellosaurus.org/CVCL_4D10 ) and the mouse brain endothelial cell line bEnd.3 (ATCC CRL-2299) were subcultured and stored in banks at −150 ° C. Cells were thawed as needed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 25 ug/mL gentamicin (Gibco), and 1X endothelial cell growth supplement (ScienCell).

Techniques:

Journal: Nature Communications

Article Title: Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2

doi: 10.1038/s41467-025-68058-9

Figure Lengend Snippet: A 1 h pretreatment with Fgf2 increases serine-9 phosphorylation of GSK3β in HBEC-5i when compared to no treatment (CTRL 0 h). TNF-α treatment induces rapid, transient dephosphorylation of GSK3β, but this effect is not reversed by Fgf2 coadministration. Each dot represents a replicate ( n = 3). B 1 h Fgf2 pretreatment diminishes basal β-catenin phosphorylation when compared to no treatment (CTRL 0 h). Further, while TNF-α induces a rapid reduction in phosphorylated β-catenin, Fgf2 reverses this dynamic upon inflammatory activation ( n = 3). C In health control endothelial cells (top), β-catenin interacts with VE-Cadherin at the cell membrane, and this complex inhibits Cldn5 transcriptional suppression by FOXO1. Excess cytosolic β-catenin is phosphorylated by GSK3β, targeting it for degradation. When stimulated with TNFα, unbound β-catenin complexes with FOXO1, leading to suppression of Cldn5 expression (bottom, red arrow), while a small amount is targeted for degradation. Meanwhile, when FGF2 is co-administered with TNF-α (bottom, blue arrow), our results suggest that unbound β-catenin is strongly redirected toward GSK3β-mediated phosphorylation. D 30 min of TNF-α is sufficient to induce β-catenin distribution at tight junctions ( n = 4 replicates) with representative images on the right ( E ) (scalebar = 20 μm). F Fgf2 attenuates TNF-α-induced reductions in the wound healing capacity of HBEC-5i ( n = 4 replicates) (**** p < 0.0001). Data represent mean ± s.e.m., and each experiment was replicated at least twice on independent samples. Group comparisons were evaluated with one or two-way ANOVA followed by Bonferroni’s post hoc tests or two-tailed t-tests with Welch’s correction when appropriate; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The human brain microvascular endothelial cell line HBEC-5i (ATCC CRL-3245, male donor according to https://www.cellosaurus.org/CVCL_4D10 ) and the mouse brain endothelial cell line bEnd.3 (ATCC CRL-2299) were subcultured and stored in banks at −150 ° C. Cells were thawed as needed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 25 ug/mL gentamicin (Gibco), and 1X endothelial cell growth supplement (ScienCell).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Activation Assay, Control, Membrane, Expressing, Two Tailed Test