cell culture mouse brain microvascular ec bend  (ATCC)

Journal: Nature Communications

Article Title: Pericytes are organ-specific regulators of tissue morphogenesis

doi: 10.1038/s41467-026-71643-1

Figure Lengend Snippet: a , b Confocal images of Bdnf iPCKO and littermate control lungs stained for ICAM2 (green) ( a) or ERG (green) and DAPI (blue) ( b ). c Confocal images of Bdnf iPCKO and littermate control lungs showing PDGFRβ-labeled pericytes (green) and DAPI-stained nuclei (blue). d Confocal images of Bdnf iPCKO and littermate control lungs showing EdU (red)-stained ECs (ERG+ , green). e Confocal images of NKX2.1-stained alveolar epithelial cells (gray) and LAMP3+ type 2 alveolar epithelial cells (green) in Bdnf iPCKO and littermate control lungs. f–m Graphs showing the ICAM2-stained vascular density based on 3D reconstruction surface images, as shown in ( a ) ( f ), percentage of ERG+ EC nuclei in total cells ( g ), percentage of PDGFRβ+ cells in total cells ( h ), ratio of ERG+ cells to PDGFRβ+ cells ( i ), the number of EdU+ ECs per area (283 × 283 × 22 µm) ( j ), ratio of LAMP3+ AT2 epithelial cells to total cells ( k ), airspace volume ( l ), and lung volume measurement ( m ) in P21 Bdnf iPCKO and littermate control lungs. Data represent mean ± s.e.m. ( n = 4 ( f – i ), n = 5 ( j–l ), n = 8 ( m ); P -values, unpaired two-tailed Student t -test. n Confocal images of Bdnf iPCKO and control lungs stained for RAGE (gray). o Confocal images showing TrkB immunostaining (gray) in ICAM2+ (red) ECs in pulmonary capillaries. p Validation of expression of Ntrk2 (encoding TrkB) in ECs by scRNA-seq analysis. UMAP plot showing color-coded EC subclusters, namely arterial, venous, general capillary (gCap) and aerocytes (aCap), in P21 lung (left). Ntrk2 expression in gCap endothelial cells (right). q Western blot showing TrkB and Phospho-TrkB (p-TrkB) in Bdnf iPCKO and littermate control total lung lysates. Molecular weight marker (kDa) is indicated. β-Actin is shown as a loading control. Quantitation of p-TrkB/TrkB ratio is shown in the graph below. Data represent mean ± s.e.m. ( n = 4 control and 3 mutants); P -values, unpaired two-tailed Welch’s test. r Confocal images of ICAM2+ (red) and ERG+ (green) endothelial cells, and PDGFRβ+ cells (gray) in EC-specific Ntrk2 iΔEC loss-of-function mutant and littermate control lungs. s Confocal images of Ntrk2 iΔEC and control lungs showing EdU (red)-stained ECs (ERG+, green).Graphs showing the ICAM2-stained vascular density ( t ), percentage of ERG + EC nuclei in total cells ( u ), percentage of PDGFRβ+ cells in total cells ( v ), ratio of ERG+ cells to PDGFRβ+ cells ( w ), the number of EdU+ ECs per area (283 × 283 × 22 µm) ( x ), airspace volume ( y ), and lung volume measurement ( z ) in P21 Ntrk2 iΔEC and littermate control lungs. Data represent mean ± s.e.m. ( n = 4 ( t – x ), n = 5 ( y ), n = 7 ( z ); P -values, unpaired two-tailed Mann–Whitney test in t and unpaired two-tailed Student t -test in ( u–z ). Source data are provided as a Source Data file.

Article Snippet: Mouse brain endothelial cells (b.End3, ATCC, cat. #CRL-2299) were cultured in DMEM (Sigma, D6546) supplemented with penicillin/streptomycin (PAA, P11-010) and 10% FCS, and kept in a humidified incubator at 37 °C, 10% CO 2 .

Techniques: Control, Staining, Labeling, Two Tailed Test, Immunostaining, Biomarker Discovery, Expressing, Western Blot, Molecular Weight, Marker, Quantitation Assay, Mutagenesis, MANN-WHITNEY

Journal: Nature Communications

Article Title: Pericytes are organ-specific regulators of tissue morphogenesis

doi: 10.1038/s41467-026-71643-1

Figure Lengend Snippet: a Tile scan of coronal sections through P12 Nodal iPCKO and littermate control brain cortex immunostained for ICAM2 (green). b Confocal images of ICAM2+ (green) and ERG+ (red) endothelial cells in Nodal iPCKO and littermate control brain. c Graph showing the ICAM2-stained vascular density. Data represent mean ± s.e.m. ( n = 8 mice per group); P -values, unpaired two-tailed Student t -test. Maximum intensity projections of P12 Nodal iPCKO and littermate control brain sections. Images show PDGFRβ+ pericytes (gray) and ERG+ (red) ECs ( d ), and EdU+ (gray) proliferating cells together with ICAM2+ (green) ECs ( e ). Graphs showing the ratio of ERG+ EC nuclei to total cells ( f ), ratio of PDGFRβ+ cells to total cells ( g ), ratio of ERG+ cells to PDGFRβ+ cells ( h ), and the number of EdU+ ECs per area (283 × 283 × 22 µm) in P12 Nodal iPCKO and littermate control brain cortex ( i ). Data represent mean ± s.e.m. ( n = 6 in f–i ); P -values, unpaired two-tailed Student t -test. j Western blot analysis of total and phosphorylated SMAD2 (p-SMAD2) in lysates of mouse brain endothelial cells (bEnd.3) treated with recombinant human Nodal (rhNodal) and SB431542 inhibitor, as indicated. Shown is a representative blot of three independent experiments. Molecular weight marker (kDa) is indicated. β-Actin is shown as a loading control. Quantitation of p-SMAD2/SMAD2 ratio is shown in the graph on the right. Data represent mean ± s.e.m. ( n = 3 independent experiments); P -values, one-way ANOVA with Tukey’s test. k Confocal images of VE-cadherin+ (CDH5, green) and EdU+ (white) bEnd.3 cells treated with rhNodal in the presence/absence of SB431542 inhibitor. l Bright field images showing that rhNODAL increases bEnd.3 cell migration in vitro in a scratch wound assay, which is blocked by SB431542. Left column shows the start of the assay (0 h) and red lines indicate the edges of scratch wounds, whereas images on the right are taken after 26 h. Graphs showing the impact of rhNodal on bEnd.3 cell proliferation ( m ) and migration ( n ). Data represent mean ± s.e.m. ( n = 6); P -values, one-way ANOVA with Tukey’s test. Source data are provided as a Source Data file.

Article Snippet: Mouse brain endothelial cells (b.End3, ATCC, cat. #CRL-2299) were cultured in DMEM (Sigma, D6546) supplemented with penicillin/streptomycin (PAA, P11-010) and 10% FCS, and kept in a humidified incubator at 37 °C, 10% CO 2 .

Techniques: Control, Staining, Two Tailed Test, Western Blot, Recombinant, Molecular Weight, Marker, Quantitation Assay, Migration, In Vitro, Scratch Wound Assay Assay

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