Journal: bioRxiv

Article Title: Uncovering the transcriptional hallmarks of endothelial cell aging via integrated single-cell analysis

doi: 10.1101/2025.08.18.669055

Figure Lengend Snippet: a. Log2 fold change (Log2FC) heatmap of marker genes related to glycosylation, per tissue and vascular bed subtype. Color scale: yellow, increased expression in aged versus young ECs; blue, decreased expression in aged versus young ECs. Asterisks indicate adjusted p-value of 0.1 or lower. b. Dot plot heatmap of predicted transcription factor (TF) activities of indicated markers involved in mechanoregulation, across tissue ECs. Color scale: yellow, high activity in aged ECs; blue, high activity in young ECs. c. Schematic of spatial transcriptomics analysis. d. Representative image of spatially resolved blood vascular (red) and lymphatic (yellow) ECs identified in human skin, as captured via MERFISH spatial transcriptomics. e. Hematoxylin and Eosin (H&E) staining of human skin tissue shown in (d). Scale = 500 μm. f. UMAP representation of human skin ECs, as captured via MERFISH spatial transcriptomics (n=2). Color-coding reflects vascular bed subtype. g-h. YAP1 (g) and TEAD1 (h) expression in human skin ECs, as captured via MERFISH spatial transcriptomics. Color scale: back, high expression; grey, low expression. i. Representative immunofluorescent images of human skin sections from young and aged subjects, immunostained for YAP (red), CD31 (green), and DAPI (blue). Smaller images are magnifications of the respective boxed areas. Arrowheads indicate nuclei with high YAP signal (within CD31 + areas). Scale = 200 μm. j. Quantification of the percentage of cells with a YAP nuclear/cytoplasmic ratio > 1 (i.e., indicative of increased mechanical signaling) in young and aged subgroups. Mean ±SEM, unpaired t-test, two-tailed, *p < 0.05, n = 10 and 8 for young and aged groups, respectively.

Article Snippet: Briefly, clearing solution was aspirated and samples washed twice using Sample Prep Wash Buffer (Vizgen, 2030001), followed by a 15-minute incubation in the DAPI and PolyT Staining Reagent (Vizgen, 20300021) on a rocker.

Techniques: Marker, Glycoproteomics, Expressing, Activity Assay, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Uncovering the transcriptional hallmarks of endothelial cell aging via integrated single-cell analysis

doi: 10.1101/2025.08.18.669055

Figure Lengend Snippet: a. UMAP representation of all HAECA ECs. Color-coding reflects CDKN1A + (purple) and CDKN1A -(grey) cell fractions. b. Principal component analysis (PCA) of early (n = 5), middle (n = 2), late passage (n = 5) HUVECs, as well as early passage HUVECs treated with doxorubicin (n = 3) as analyzed by bulk RNA-sequencing; projection along the two first principal components is shown. c. Bar graph visualization of top 5 most enriched GO terms in early (left) and late (right) passage HUVECs. d. Heatmap visualization of representative genes enriched at different passage numbers/ conditions of induced cellular senescence. Dashed lines indicate gene clusters associated with the indicated conditions. Color scale: red, high expression; blue, low expression. e. Representative images of p21 (magenta), SPiDER-β-gal staining (green) and DAPI (blue) immunofluorescence staining in early and late passage HUVECs, as well as Doxorubicin-induced (Dox) HUVECs. Scale is 100 μm. f. Quantification of nuclear p21 staining intensity (left) and SPiDER-β-gal signal intensity (right) in early passage, late passage, and Doxorubicin treated HUVECs (n = 7 each). Mean ±SD, *p < 0.05, by one-way ANOVA. g. Schematic of CDKN1A -associated signature analysis. h. Violin plots of the expression of pan-tissue senescence-associated markers in each tissue. Data is split between CDKN1A + (purple) and CDKN1A -(grey) subgroups. i. Upper panel: schematic of induced pluripotent stem cells (iPSC) to induced brain-like EC (iBEC) differentiation, and subsequent induction of replicative senescence. Lower panel: differential gene expression analysis between early (n=3) and late passage (n=4) iBECs, for the indicated pan-tissue senescence markers. Wald test, *p < 0.05, **p < 0.01 (adjusted p-values). j. Scatterplot of normalized expression of Cdkn1a (x-axis) and Maff (y-axis) in EC-WT (left) and EC-Prog-Tg (right). Expression was normalized to housekeeping gene Hprt . R values are indicated in each plot. n=3 for EC-WT, n=4 for EC-Prog-Tg for each indicated tissue (heart and liver).

Article Snippet: Briefly, clearing solution was aspirated and samples washed twice using Sample Prep Wash Buffer (Vizgen, 2030001), followed by a 15-minute incubation in the DAPI and PolyT Staining Reagent (Vizgen, 20300021) on a rocker.

Techniques: RNA Sequencing, Expressing, Staining, Immunofluorescence, Gene Expression

Journal: bioRxiv

Article Title: Uncovering the transcriptional hallmarks of endothelial cell aging via integrated single-cell analysis

doi: 10.1101/2025.08.18.669055

Figure Lengend Snippet: a. Schematic of SenMayo-associated analysis. b. Violin plots of the expression of pan-tissue senescence-associated markers in each tissue. Data is split between SenMayo high (top 25%, dark blue), SenMayo middle (middle 50%, light blue), and SenMayo low (bottom 25%, grey) subgroups. c. Schematic of CellAge(ind)-associated analysis. d. Violin plots of the expression of pan-tissue senescence-associated markers in each tissue. Data is split between CellAge high (top 25%, dark green), CellAge middle (middle 50%, light green), and CellAge low (bottom 25%, grey) subgroups. e. Volcano plot of differentially expressed genes between iBECs (right) and non-iBECs (left, iECs and HUVECs). Significant genes (adj. p-value < 0.05 and Log2 fold change > 1) are shown in orange. f. Representative images of CLDN5 (top) or GLUT1 (bottom) immunofluorescence staining in early passage iBECs. Nuclei are stained with DAPI (blue). Scale is 100µm. g. Dot plot visualization of iBEC-specific marker genes in the HAECA dataset (stratified by tissue). The color intensity of each dot represents the average level of marker expression, the dot size reflects the percentage of ECs expressing the marker within each tissue. Color scale: yellow, high expression; blue, low expression. h. Normalized mRNA expression values of the indicated marker genes in aortic ECs derived from WT and Lmna G609G/G609G mice (scRNA-seq dataset derived from ). n=2 for WT and Lmna G609G/G609G groups. i. Genotyping PCR for tetop-LAG608G (961 bp, top) and Cdh5-tTa (461 bp, bottom). Lower bands (∼200 bp) are internal amplification controls. Samples positive for both transgenes are classified as mutant. Samples positive for only one or neither transgene are classified as wild type. Lane 1: molecular weight DNA ladder. The last two lanes represent the positive and negative controls, respectively.

Article Snippet: Briefly, clearing solution was aspirated and samples washed twice using Sample Prep Wash Buffer (Vizgen, 2030001), followed by a 15-minute incubation in the DAPI and PolyT Staining Reagent (Vizgen, 20300021) on a rocker.

Techniques: Expressing, Immunofluorescence, Staining, Marker, Derivative Assay, Amplification, Mutagenesis, Molecular Weight