Journal: bioRxiv

Article Title: Endothelial cell derived microvesicles modulate cerebrovascular and brain aging via miR-17-5p and serve as potential biomarkers for vascular aging

doi: 10.1101/2023.02.02.526751

Figure Lengend Snippet: (A) Immunofluorescent characterization of BMECs. The isolated BMECs were 95% positive for CD31(red), confirming the epithelial nature of these cells (scale bar=30 μm, n = 3 technical replicates). (B) Identification of senescence of BMECs analyzed by SA-β-gal staining (scale bar=200 μm). Senescent BMECs showed higher percentage of SA-β-gal positive stained cells marked blue (>60%) than young BMECs (Student’s t -test, mean ± SEM, n = 6 technical replicates, ** p < 0.01). (C) Representative transmission electron microscopy (TEM) analysis of y.EMVs/o.EMVs and y.EEXs/o.EEXs (scale bar=200 nm for EMVs, scale bar=100 nm for EEXs, n = 3 technical replicates). (D) Average size distribution and concentration of y.EMVs/o.EMVs and y.EEXs/o.EEXs determined by nanoparticle tracking assay ( n = 3 technical replicates). (E) Confocal microscopy images of the internalization of EMVs and EEXs labeled with PKH 26 (marked red) in mouse brain vasculature, nuclei were stained with DAPI (marked blue), blood vessels were stained with CD31 + (marked green), scale bar=20 μm, n = 6 biological replicates per group. (F) Confocal microscopy images of the internalization of EMVs labeled with PKH 26 (marked red) in BMECs, nuclei were stained with DAPI (marked blue), cell membrane was stained with DIO (marked green), scale bar=20 μm. n = 3 technical replicates.

Article Snippet: Finally, DAPI (1 μM, Beyotime, cat# C1006) was used for staining cellular nuclear in BMECs and brain slices.

Techniques: Isolation, Staining, Transmission Assay, Electron Microscopy, Concentration Assay, Confocal Microscopy, Labeling, Membrane

Journal: bioRxiv

Article Title: Endothelial cell derived microvesicles modulate cerebrovascular and brain aging via miR-17-5p and serve as potential biomarkers for vascular aging

doi: 10.1101/2023.02.02.526751

Figure Lengend Snippet: (A, B) Representative images of SA-β-gal staining in basilar artery (Scale bar=20 μm, n = 6 biological replicates per group). (C, D) Representative images of CBF tested by laser doppler flowmetry in diverse groups ( n = 10 biological replicates per group). (E, F) Confocal microscopy images of cerebral microvascular density marked with CD31 + in mice brain. Red, CD31 + ; blue, DAPI for nuclei staining (scale bar=50 μm, n = 6 biological replicates per group). (G) The percentage of SA-β-gal staining positive cell in basilar artery (one-way ANOVA, Tukey’s multiple comparisons test, mean ± SEM, n =6 biological replicates per group, * p < 0.05, *** p < 0.001). (H) Quantification of CBF in various groups (Kruskal–Wallis ANOVA, post hoc Dunn’s multiple comparison test, mean ± SEM, n = 10 biological replicates per group, * p < 0.05, *** p < 0.001). (I) Quantification of vessel density in various groups (one-way ANOVA, Tukey’s multiple comparisons test, mean ±SEM, n = 6 biological replicates per group, * p < 0.05, ** p < 0.01, *** p < 0.001). (J) EB extravasation in mice brains (one-way ANOVA, Tukey’s multiple comparisons test, mean ± SEM, n = 4 biological replicates per group, experiment repeated for 3 times, * p < 0.05, ** p < 0.01, *** p < 0.001). (K, L) Representative images of SA-β-gal positive cells in CA3 region of hippocampus in diverse groups (scale bar=100 μm. n = 6 biological replicates per group). (M) Quantification of SA-β-gal positive cell in various groups (one-way ANOVA, Tukey’s multiple comparisons test, mean ± SEM, n = 6 biological replicates per group, * p < 0.05, ** p < 0.01, *** p < 0.001). (N) Plasma GSSG expression of mice in diverse groups (one-way ANOVA, Tukey’s multiple comparisons test, mean ± SEM, n = 6 biological replicates per group, * p < 0.05, ** p < 0.01, *** p < 0.001). (O-Q) MDA, TERT and klotho expression in mice brain (one-way ANOVA, Tukey’s multiple comparisons test, mean ± SEM, n = 6 biological replicates per group, * p < 0.05, ** p < 0.01, *** p < 0.001, ns , no significance).

Article Snippet: Finally, DAPI (1 μM, Beyotime, cat# C1006) was used for staining cellular nuclear in BMECs and brain slices.

Techniques: Staining, Confocal Microscopy, Comparison, Clinical Proteomics, Expressing

Journal: bioRxiv

Article Title: Endothelial cell derived microvesicles modulate cerebrovascular and brain aging via miR-17-5p and serve as potential biomarkers for vascular aging

doi: 10.1101/2023.02.02.526751

Figure Lengend Snippet: (A, B) Representative images of co-staining of CD31 + (marked red) with ROS (marked green) in various groups. CD31 + is accepted as endothelium marker, DAPI (marked blue) for nuclei staining (scale bar=30 μm. n = 6 biological replicates per group). (C) Summary of ROS expression in mouse brain vasculature (one-way ANOVA, Tukey’s multiple comparisons test, mean ±SEM, n = 6 biological replicates per group, * p < 0.05, ** p < 0.01, *** p < 0.001). (D, E) Immunofluorescent co-staining of CD31 + (marked red) with TUNEL (marked green) in mouse brain vasculature in various groups. TUNEL is regarded as apoptosis marker (scale bar=30 μm. n =6 biological replicates per group). (F) Summary of apoptotic levels in mouse brain vasculature (one-way ANOVA, Tukey’s multiple comparisons test, mean ± SEM, n = 6 biological replicates per group, * p < 0.05, ** p < 0.01, *** p < 0.001). (G, H) Immunofluorescent co-staining of CD31 + (marked red) with NO (marked green) in various groups (scale bar=30 μm. n = 6 biological replicates per group). (I) Summary of NO expression in mouse brain vasculature (one-way ANOVA, Tukey’s multiple comparisons test, mean ± SEM, n = 6 biological replicates per group, ** p < 0.01, *** p < 0.001). Data information: For (A-I), same threshold was applied over all pictures.

Article Snippet: Finally, DAPI (1 μM, Beyotime, cat# C1006) was used for staining cellular nuclear in BMECs and brain slices.

Techniques: Staining, Marker, Expressing, TUNEL Assay

Journal: Breast Cancer Research : BCR

Article Title: BRCA1 mutation influences progesterone response in human benign mammary organoids

doi: 10.1186/s13058-019-1214-0

Figure Lengend Snippet: Generation of human mammary organoids. a Top, Schematic of overall study design is shown. Bottom panel is the 28-day stepwise menstrual cycle hormone profile. Day 0–day 14 Follicular phase, day 14–day 28 luteal phase. b PR and ER were co-stained with luminal marker (CK18) in BRCA1 mut and non-carrier organoids. Immunofluorescent staining was done for ER (red) and PR (red) along with CK18 (green) and DAPI (blue) to visualize the nuclei. Scale bar, 100 μm. Co-localization is shown by red arrows. c Normalized expression of ER and PR mRNA in BRCA1 mut and non-carrier breast organoids is shown. Unpaired t -test was performed

Article Snippet: Nuclei were counter stained with either hematoxylin for 30 s or 1 μM DAPI (Invitrogen, D1306) for 15 min. For collagen staining, a trichrome stain kit (Abcam, ab150686) was used according to the manufacturer’s instructions.

Techniques: Staining, Marker, Expressing

Journal: Breast Cancer Research : BCR

Article Title: BRCA1 mutation influences progesterone response in human benign mammary organoids

doi: 10.1186/s13058-019-1214-0

Figure Lengend Snippet: Expression of ALDH1 and cell type-specific genes in BRCA1 mut and non-carrier mammary organoids . a Immunofluorescent staining was done in BRCA1 mut and non-carrier organoids with ALDH1 (red) and DAPI (blue) to visualize the nuclei. Scale bar, 100 μm. b Normalized expression of ALDH1 mRNA in individual BRCA1 mut and non-carrier mammary organoids is shown ( N = 4, p = 0.1106, unpaired t -test). c–f Normalized reads in individual BRCA1 mut and non-carrier organoids treated with E2+P4 are shown in the left panels for c luminal HR-positive genes, d luminal HR-negative genes, e myoepithelial genes, and f fibroblasts genes. The effect of TPA treatment in BRCA1 mut and non-carrier organoids are shown as fold changes of hormone treatment (red dotted line = 1) in the right panels for each cell-specific genes (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Nuclei were counter stained with either hematoxylin for 30 s or 1 μM DAPI (Invitrogen, D1306) for 15 min. For collagen staining, a trichrome stain kit (Abcam, ab150686) was used according to the manufacturer’s instructions.

Techniques: Expressing, Staining