dermal microvascular endothelial cells mecs  (PromoCell)

Journal: Cell Death & Disease

Article Title: Pharmacological activation of p53 induces dose-dependent changes in endothelial cell fate during angiogenic sprouting

doi: 10.1038/s41419-025-08292-7

Figure Lengend Snippet: A Concentration-dependent growth inhibition of human umbilical vein endothelial cells (HUVEC) by three p53-activators (MDM2 inhibitors navtemadlin and nutlin-3a; MDM2/MDMX inhibitor sulanemadlin), but not by non-specific control peptide. Cell growth was measured by live-cell imaging as the percent confluence normalized to untreated wells following 72 h treatment. Data points show averaged value from one experiment ( n = 3 experiments) and are fitted with a best fit model for concentration-growth response. B Growth inhibition of human dermal microvascular endothelial cells (HDMEC) and normal human dermal fibroblasts (NHDF) by navtemadlin, as measured by live-cell imaging. Data points show averaged value from one experiment ( n = 3 experiments) and are fitted with a best fit model for concentration-growth response. C Growth inhibition of HUVEC recovers within 48 h following an initial 24 h treatment using ≤ 0.1 μM navtemadlin. Cell growth was measured by live-cell imaging and quantified over 72 h as cell counts per well and normalized to counts at time 0. Data points show mean ± SD ( n = 3 independent experiments). * P adj < 0.05, ** P adj < 0.01 using 1-way, repeated measures ANOVA with adjustment using Dunnett’s correction. D Morphological abnormalities are visible in phase-contrast images of venous ECs (HUVEC) and capillary ECs (HDMEC), but not in those of fibroblasts (NHDF) after 72 h of navtemadlin treatment, but not after 24 h. Scale bar = 200 μm. E Increased expression of proteins involved in p53 signaling (MDM2, clone IF2, 0.5 μg/mL; p53, clone DO-1, 0.4 μg/mL), cell cycle arrest (p21, clone 12D1, 0.24 μg/mL), and apoptosis (PUMA, clone D30C10, 0.96 μg/mL) in HUVEC following 24 h navtemadlin treatment, as determined by western blot analysis. Total protein controls correspond to distinct membranes (L1 or L2). Images are cropped from full-length blots of one biological experiment (see ‘Full Length Western Blots’) and are representative of at least two experiments. Further details regarding antibodies used are shown in SI Table . F Increased expression of p53 (clone DO-1, 12 μg/mL), cell cycle arrest (p21, clone 12D1, 1.22 μg/mL), apoptosis (PUMA, clone D30C10, 4.8 μg/mL), dead cells (Sytox green, 100 nM), and senescence (β-galactosidase), and reduced expression of active cell cycle (Ki67, clone SP6, 0.12 μg/mL), following 24 h treatment of HUVEC using navtemadlin as visualized by immunofluorescence, live-cell fluorescence imaging, and colorimetric staining. Scale bar = 50 μm. Further details regarding antibodies and concentrations used are shown in SI Table . G –K Quantification of fluorescence and colorimetric levels of protein markers, showing increased expression of p53, cell cycle arrest (p21), apoptosis (PUMA), cell death (Sytox green), and senescence, and reduced activity in cell cycle (Ki67). Data points indicate value from one experiment ( n = 3 experiments). ** P adj < 0.01, *** P adj < 0.001 using one-way ANOVA with adjustment using Dunnett’s correction. Horizontal black line indicates the mean value.

Article Snippet: For in vitro assays, we used commercially available primary cultures of three cell lines: human umbilical vein endothelial cells (HUVEC) from pooled donors (cat # C-12203, Promocell), human dermal microvascular endothelial cells (HDMEC) from adult donors (cat # C-12212, Promocell), and normal human dermal fibroblasts (NHDF) from adult donors (cat # 106-05 A, Cell Applications Inc).

Techniques: Concentration Assay, Inhibition, Control, Live Cell Imaging, Expressing, Western Blot, Immunofluorescence, Fluorescence, Imaging, Staining, Activity Assay