Journal: bioRxiv

Article Title: Endothelial expression of ZBTB16 protects against cardiac aging

doi: 10.1101/2025.10.08.681100

Figure Lengend Snippet: ( A ) siRNA mediated knockdown of ZBTB16 in human umbilical vein endothelial cells (HUVEC). Gene silencing was confirmed 72h after siRNA transfection (n=6). ( B ) Network formation assay of HUVEC transfected with siCtrl or siZBTB16 (n=4). ( C ) Quantification of Boyden Chamber transmigration assay using HUVEC after siCtrl Vs. siZBTB16 transfection. Data are normalized to siCtrl-treated cells without VEGFA. ( D ) Spheroid sprouting assay of HUVEC transfected with siCtrl or siZBTB16 followed by treatment with or without VEGFA (n=4 and n=5). Representative images are shown in the left panel. ( E ) Cell numbers were counted by visual assessment after 72h of knockdown (n=5). ( F ) Quantification of Annexin V and 7-AAD FACS. (n=3). ( G ) Representative images of senescence associated-β-galactosidase staining (senescence) at 72 h of siRNA treatment. Quantification is shown in the right panel (n=5). Data are shown as mean and error bar indicate the standard error of the mean. P-value was calculated by two-tailed Student’s t-test.

Article Snippet: Human umbilical cord vein endothelial cells (HUVEC) were purchased from PromoCell (C-122203) and cultured in endothelial basal medium (EBM, CC-3121, Lonza) supplemented with 10% fetal bovine serum (10100147, Gibco).

Techniques: Knockdown, Transfection, Tube Formation Assay, Transmigration Assay, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Endothelial expression of ZBTB16 protects against cardiac aging

doi: 10.1101/2025.10.08.681100

Figure Lengend Snippet: ( A ) Bulk RNA sequencing of ZBTB16 knockdown cells and siRNA control cells (n=5). Graph shows log2 fold-change of genes associated with senescence (based on SenMayo (PMID: 35974106)). Genes were significant (Bonferroni adjusted p-value < 0.05). Positive log2 fold-change indicates higher expression in ZBTB16 knockdown cells. Genes were annotated according to their biological function. ( B-D ). Fibroblasts treated 72h with supernatants of HUVEC, which were transfected with siCtrl or siZBTB16 for 72h (n=10). Arrows indicate COL1A1 expression. Quantification is shown in c for COL1A1 (72h) and d for aSMA (48h). ( E-F ) Collagen gel contraction assay at baseline (upper panel) and after 96h (bottom panel) of fibroblast gels treated with supernatants from siCtrl (left) or siZBTB16 (right). Red line indicates gel boundaries. Quantification of relative decrease in gel area is shown in F. ( G-I ) Cardiac tissue mimetics (CTMs) containing primary rat cardiomyocytes, fibroblasts and endothelial cells were treated with supernatants of siCtrl or siZBTB16-transfected HUVEC for 14d. Representative immunohistochemical stainings are shown in G. Quantification of alpha smooth muscle actinin (aSMA) (H) and collagen COL1A1 (I). ( J-L ) siCtrl or siZBTB16-transfected HUVEC were directly co-cultured with neonatal rat cardiomyocytes (n=8) for 96h and stained for DAPI (blue), Phalloidin (green), sarcomeric-actinin (red) and VE-cadherin (magenta). Arrows indicate cardiomyocytes depicted by high sarcomeric actinin content. Cardiomyocyte hypertrophy quantified in panel k, and cardiomyocyte beating frequency is shown in L. ( M-O ) Neonatal rat cardiomyocytes were cultured in the supernatant of HUVEC after siCtrl or siZBTB16 transfection. Contraction (peak time) and relaxation (return velocity time, return to baseline 90%) were determined using IonOptix. Single cardiomyocytes were analyzed in the presence and absence of phenylephrine (-PE: n=19 vs. n=22; +PE: n=22 vs. n=23). ( P ) Primary mouse cortical neurons were treated with supernatants of siCtrl or siZBTB16-transfected HUVEC. Quantification is shown in the right panel. ( Q ) Innervation as assessed histologically by TUJ1 (green) normalized to IB4 (red) in hearts of Zbtb16 +/+ and Zbtb16 +/- mice (n=6 vs. n=7). Quantification is shown in the right panel. Data are shown as mean and error bar indicate the standard error of the mean. P-value was calculated by two-tailed Student’s t-test.

Article Snippet: Human umbilical cord vein endothelial cells (HUVEC) were purchased from PromoCell (C-122203) and cultured in endothelial basal medium (EBM, CC-3121, Lonza) supplemented with 10% fetal bovine serum (10100147, Gibco).

Techniques: RNA Sequencing, Knockdown, Control, Expressing, Transfection, Collagen Gel Contraction Assay, Immunohistochemical staining, Cell Culture, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Endothelial expression of ZBTB16 protects against cardiac aging

doi: 10.1101/2025.10.08.681100

Figure Lengend Snippet: ( A-C ) ZBTB16 was overexpressed by lentiviral vectors in long-term passaged HUVEC (>P9) for >8 days. (A) Staining of HUVEC for acidic-β-galactosidase (n=5). (B) ZBTB16 was overexpressed in long-term passaged senescent HUVEC (>P12) prior to performing a network formation assay (n=4). (C) ZBTB16 was overexpressed in long-term passaged HUVEC (>P12). Supernatants were collected and transferred to human cardiac fibroblasts (n=5). After 72h, fibroblasts were stained for COL1A1 (grey), DAPI (blue), aSMA (red) and Phalloidin (green). Scale bar indicates 50 µm. ( D-I ) Overexpression of Zbtb16 in endothelial cells by targeted AAV9 vectors improves cardiac function in 18-month-old mice. (D) Zbtb16 expression in liver endothelial cells 4 weeks after AAV9 treatment. (E) Diastolic heart function and F ejection fraction 8 weeks after AAV9 treatment. Acidic β-galactosidase (G) of cardiac sections 8 weeks after AAV9 treatment. (H, I) Vascular fibrosis as assesses by Sirius red staining on heart section from 3-month-old (n=3), 18-month-old (n=3), 20-month-old (n=5) and 20-month-old mice after two months of ZBTB16-AAV9 or control treatment (n=6). Data are shown as mean and error bar indicate the standard error of the mean. P-value was calculated by two-tailed Student’s t-test or using One-way ANOVA with a post-hoc Tukey’s test (H).

Article Snippet: Human umbilical cord vein endothelial cells (HUVEC) were purchased from PromoCell (C-122203) and cultured in endothelial basal medium (EBM, CC-3121, Lonza) supplemented with 10% fetal bovine serum (10100147, Gibco).

Techniques: Staining, Tube Formation Assay, Over Expression, Expressing, Control, Two Tailed Test

Journal: Advanced Science

Article Title: Versatile DNA Hydrogel‐Mediated Delivery of Ginsenoside‐Encapsulated Small Extracellular Vesicles to Boost Diabetic Wound Repair

doi: 10.1002/advs.202522920

Figure Lengend Snippet: GS/sEV in vitro Promotion on cell migration and angiogenesis. (A,B) Cellular uptake of GS/sEV complexes by HaCaT cells within 4 h, detected by laser scanning confocal microscope (red: PKH26‐labeled GS/sEV; blue: DAPI nuclei staining; scale bar: 20 µm). (C,D) Flow cytometry analysis of GS/sEV internalization mechanism, revealing that lipid raft‐dependent endocytosis is the predominant uptake pathway. (E) Viability of the treated HaCaT cells assessed by CCK‐8 assay. (F,G) Transwell assays on HaCaT cell migration at 12, 24, and 48 h (representative images and quantified migration rates; scale bar: 200 µm). (H,I) Tube formation assay of HUVECs on Matrigel (representative images and branch quantification; scale bar: 200 µm). (J) Wound healing assay for HaCaT cells observed under microscope at 12, 24, and 48 h (scale bar: 200 µm). (K,L) VEGFA protein expression in HUVECs was measured by Western blot assay, band intensities were quantified using ImageJ software, and the expression of VEGFA was normalized to β‐actin. (M) The relative VEGFA mRNA levels in HUVECs measured by RT‐qPCR (normalized to GAPDH; n = 3). Data were presented as mean ± SD, and statistical significance was calculated by one‐way ANOVA with Tukey's multiple comparisons test.

Article Snippet: Mesenchymal stem cells (MSCs), human umbilical cord endothelial cells (HUVECs), human immortalized epidermal cells (HaCaT), mouse macrophage cells (RAW264.7), human kidney proximal tubular cells (HK‐2), and human retinal müller cells were obtained from Procell Biotechnology Co., Ltd. (Wuhan, China).

Techniques: In Vitro, Migration, Microscopy, Labeling, Staining, Flow Cytometry, CCK-8 Assay, Tube Formation Assay, Wound Healing Assay, Expressing, Western Blot, Software, Quantitative RT-PCR