mouse brain microvascular endothelial cell line bend 3  (ATCC)

Journal: Open Life Sciences

Article Title: Doxorubicin compromises blood-brain barrier integrity by suppressing annexin A1 expression

doi: 10.1515/biol-2025-1297

Figure Lengend Snippet: Effects of DOX, miR-196a antagonist, and AP-1 inhibitor on blood-brain barrier (BBB) integrity, tight junction proteins, and annexin A1 expression. A: Quantitative analysis of Evans blue (EB) extravasation (μg/g tissue), occludin, and claudin-5 protein expression in the hippocampus of mice. Data are presented as the mean ± standard deviation (SD). Protein expression was normalized to GAPDH. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001 versus Control group (group C); # P < 0.05, ## P < 0.01, ### P < 0.001 versus DOX-treated group (group D); ns, not significant (adjusted P > 0.05). B: Representative Western blot bands of occludin and claudin-5 proteins in mouse hippocampal tissues. GAPDH served as the loading control. Bands were selected from three independent experiments with high reproducibility, and integrated optical density (IOD) was quantified using ImageJ software. C: Immunofluorescence staining of annexin A1 (ANXA1) in bEnd.3 cells. Green fluorescence indicates ANXA1-positive signals, and blue fluorescence indicates DAPI-stained nuclei. Scale bar = 300 μm. All images were captured with identical exposure parameters. D: Quantitative analysis of the ANXA1-positive area per cell (μm 2 ) in bEnd.3 cells. Data are presented as the mean ± SD Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001 versus Group C; # P < 0.05, ## P < 0.01, ### P < 0.001 versus Group D; ns, not significant (adjusted P > 0.05).

Article Snippet: The mouse brain microvascular endothelial cell line bEnd.3 (American Type Culture Collection, ATCC ® CRL-2299TM) was utilized in this study as an in vitro model of the blood-brain barrier.

Techniques: Expressing, Standard Deviation, Control, Western Blot, Software, Immunofluorescence, Staining, Fluorescence

Journal: Open Life Sciences

Article Title: Doxorubicin compromises blood-brain barrier integrity by suppressing annexin A1 expression

doi: 10.1515/biol-2025-1297

Figure Lengend Snippet: miR-196a directly targets annexin A1 (ANXA1) and is upregulated by DOX. A: Quantitative analysis of miR-196a relative expression in bEnd.3 cells. U6 snRNA was used as the internal reference, and expression levels were calculated using the 2 − ΔΔCt method. Experimental groups were consistent with Figure 1 . Data are presented as the mean ± SD Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. * P < 0.05 versus Group C; # P < 0.05 versus Group D; ns, not significant (adjusted P > 0.05). B: Schematic diagram of the predicted binding site between miR-196a-5p and the 3′ untranslated region (3′UTR) of the ANXA1 gene, including the wild-type (WT) ANXA1 3′UTR (containing the intact miR-196a-5p binding sequence) and the mutant (Mut) ANXA1 3′UTR (with site-directed mutations in the binding sequence to disrupt miR-196a-5p binding). C: Relative luciferase activity detected by dual-luciferase reporter assay in bEnd.3 cells. Cells were cotransfected with miR-196a mimics/negative control and WT/Mut ANXA1 3′UTR reporter plasmids. Data are presented as the mean ± SD Statistical analysis was performed using an unpaired two-tailed Student’s t -test. * P < 0.05 indicates a statistically significant difference.

Article Snippet: The mouse brain microvascular endothelial cell line bEnd.3 (American Type Culture Collection, ATCC ® CRL-2299TM) was utilized in this study as an in vitro model of the blood-brain barrier.

Techniques: Expressing, Binding Assay, Sequencing, Mutagenesis, Luciferase, Activity Assay, Reporter Assay, Negative Control, Two Tailed Test

Journal: Open Life Sciences

Article Title: Doxorubicin compromises blood-brain barrier integrity by suppressing annexin A1 expression

doi: 10.1515/biol-2025-1297

Figure Lengend Snippet: DOX activates AP-1 transcriptional activity in bEnd.3 cells. A: Representative Western blot bands of c-fos and c-jun proteins in bEnd.3 cells from the control group (group C) and DOX-treated group (group D). GAPDH served as the loading control. Each group includes results from two biological replicates. B: Representative Western blot bands of phosphorylated c-fos (p-c-fos) and total c-fos proteins in bEnd.3 cells from group C and group D, clearly showing the relative expression levels of the target proteins. C: Representative Western blot bands of phosphorylated c-jun (p-c-Jun) and total c-jun proteins in bEnd.3 cells from group C and group D, clearly showing the phosphorylation modification levels of the target proteins. D: Quantitative analysis of c-fos and c-jun protein relative expression in bEnd.3 cells from group C and group D. Expression levels were normalized to GAPDH. Data are presented as the mean ± SD Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. * P < 0.05 versus Group C; # P < 0.05 versus Group D. E: Quantitative analysis of the phosphorylation ratios of p-c-fos/c-fos and p-c-Jun/c-Jun proteins in bEnd.3 cells from group C and group D. Data are presented as the mean ± SD Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. * P < 0.05 versus Group C; # P < 0.05 versus Group D.

Article Snippet: The mouse brain microvascular endothelial cell line bEnd.3 (American Type Culture Collection, ATCC ® CRL-2299TM) was utilized in this study as an in vitro model of the blood-brain barrier.

Techniques: Activity Assay, Western Blot, Control, Expressing, Phospho-proteomics, Modification

Journal: bioRxiv

Article Title: Using atorvastatin-induced vascular weakness to model brain haemorrhage in vascularised cerebral organoids

doi: 10.64898/2026.04.20.719465

Figure Lengend Snippet: ( A ) Phase contrast images of HBMECs after 24 hours of treatment with 100 μM atorvastatin (ATV). Scale bars 50 μm. ( B ) Cell survival after ATV treatment for 24 hours in both stationary and rotating culture conditions ( n =4 independent repeats). Scale bars 100 μm. ( C ) qPCR analysis of marker expression after ATV treatment in rotating cells shows an increase in VE-Cadherin and NG2 gene expression (non-significant, One-Way ANOVA). ( D ) Fluorescent images of CD31 (green) and ZO-1 (red) cellular expression after 24 hours with ATV and DMSO control. ( E ) Total expression of ZO-1 after treatment ( n =4 independent repeats, two-way ANOVA *** P =0.0009, **** P <0.0001) ( F ) Analysis of ZO-1 co-localised with CD31 on the cell surface when treated with ATV ( n =4 independent repeats, two-way ANOVA, *** P =0.0002). ( G ) Fluorescent images of CD31 (green) and VE-Cadherin (red) cellular expression after 24 hours of ATV treatment in both stationary (top panels) and rotating culture (bottom panels). Zoom inset shows the internalisation of both CD31 and VE-Cadherin from the cell surface when treated with ATV. Scale bars 20 μm. ( H ) Analysis of VE-Cadherin co-localised with CD31 on the cell surface when treated with ATV ( n =3 independent repeats, two-way ANOVA, * P <0.04). ( I ) Total area expression of CD31 and VE-Cadherin as a percentage area of DAPI in both stationary and rotating cells, ( n =3 independent repeats, no significance from analysis with a two-way ANOVA).

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Innoprot P10361) were maintained on 1% gelatin coated flasks in endothelial cell growth medium MV (PromoCell C-22020).

Techniques: Marker, Expressing, Gene Expression, Control

Journal: bioRxiv

Article Title: Using atorvastatin-induced vascular weakness to model brain haemorrhage in vascularised cerebral organoids

doi: 10.64898/2026.04.20.719465

Figure Lengend Snippet: ( A ) Representative images of filipin-stained HBMECs, after 24hours of drug treatment. Scale bars 50 μm. ( B ) Filipin expression as a percentage of area from 6 ROIs in 2 wells across 2 independent repeats after 24 hours with ATV, ** P =0.0081, t -test. ( C ) qPCR analysis of rotated HBMECs after 24 hours of drug treatment shows a significant increase in HMGCR expression ( n =5 independent repeats, * P =0.0323 One-Way ANOVA).

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Innoprot P10361) were maintained on 1% gelatin coated flasks in endothelial cell growth medium MV (PromoCell C-22020).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Using atorvastatin-induced vascular weakness to model brain haemorrhage in vascularised cerebral organoids

doi: 10.64898/2026.04.20.719465

Figure Lengend Snippet: ( A ) Phase images of tube formation in untreated and ATV-treated HBMECs. Scale bars 20 μm. ( B ) Analysis of the network parameter average vessel length shows a significant reduction with ATV treatment, both pre-tube formation and post (One-way ANOVA with Tukey’s post hoc multiple comparisons test ** P =0.003, *** P =0.0007, **** P <0.0001). ( C ) Cytotoxicity analysis of ATV treated tubes showed no difference from controls ( n =3, t -test).

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Innoprot P10361) were maintained on 1% gelatin coated flasks in endothelial cell growth medium MV (PromoCell C-22020).

Techniques:

Journal: bioRxiv

Article Title: Using atorvastatin-induced vascular weakness to model brain haemorrhage in vascularised cerebral organoids

doi: 10.64898/2026.04.20.719465

Figure Lengend Snippet: ( A ) Representative confocal images of whole organoids treated with ATV for 24 hours and the loss of VE-Cadherin expression from the surface. Scale bars 500 μm. ( B ) VE-Cadherin, not CD31 ( P =0.091), expressed as a percentage of DAPI was significantly reduced with ATV treatment compared to DMSO controls (two-tailed t -test, * P =0.011). ( C ) Size progression for 4 batches of organoids that were treated with ATV at day 40. ( D ) Vascular metrics were unchanged for CD31 ( n =15 organoids from 4 batches, non-significant t- test) with slightly more endpoints, signifying single cells. ( E ) Angiotool analysis of VE-Cadherin staining revealed shorter overall vessel length ( n =15 organoids from 4 batches, t- test, * P =0.0389)). ( F ) qPCR analysis of ATV-treated organoids showed a reduction in VE-Cadherin RNA expression; however, other markers of endothelial function are unchanged (One-Way ANOVA, n =5 organoids from 2 batches). ( G ) ATV treatment increased the expression of some cholesterol biosynthesis markers compared to DMSO controls, opposite to what was observed in HBMECs in 2D (One-Way ANOVA, n =5 organoids from 2 batches).

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Innoprot P10361) were maintained on 1% gelatin coated flasks in endothelial cell growth medium MV (PromoCell C-22020).

Techniques: Expressing, Two Tailed Test, Staining, RNA Expression