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