Journal: Development (Cambridge, England)

Article Title: Endothelial cell SMAD6 balances Alk1 function to regulate adherens junctions and hepatic vascular development.

doi: 10.1242/dev.201811

Figure Lengend Snippet: Fig. 9. SMAD6 modulates endothelial cell hypercontractility and PI3K signaling via ALK1. (A-F) HUVEC treated with non-targeting (NT), Smad6-1 and/or Alk1 siRNA were cultured in static conditions on biotinylated fibronectin and treated as indicated for 15 min (thrombin, blebbistatin) (A-C) or 21 h (740-P, wortmannin) (D-F) at 37°C. (A) VE- cadherin stain. (B) Streptavidin-488 label. (C) Quantification of streptavidin-488 area/ field of view (FOV). Data are mean±s.d. (1 data point/FOV), n=3 experimental replicates/condition. Relevant subset of significant comparisons shown. (D) VE- cadherin stain. (E) Streptavidin-488 label. (F) Quantification of streptavidin-488 area/ FOV. Data are mean±s.d. (1 data point/ FOV), n=3 experimental replicates per condition. Relevant subset of significant comparisons shown. *P<0.05; **P<0.01; ****P<0.0001; ns, not significant. One-way ANOVA with Tukey’s multiple comparisons test. Scale bars: 20 µm (A,D); 100 µm (B,E).

Article Snippet: For contractility assays, HUVEC were treated with 0.5 U/ml thrombin (Sigma-Aldrich, T7201-500UN) at 37°C for 15 min. For contractility inhibition assays, HUVEC were treated with 10 μM blebbistatin (SigmaAldrich, B0560-1MG) at 37°C for 15 min. For PI3K activation assays, HUVEC were treated with 20 μM 740Y-P (MedChemExpress, HY-P0175) at 37°C for 22 h. For PI3K inhibition assays, HUVEC were treated with 100 nM wortmannin (SelleckChem, S2758) at 37°C for 22 h. For BMP9 ligand assays, HUVECwere serum starved in Endothelial BaseMedia (Lonza CC-3162) with 0.1% fetal bovine serum (FBS) for 24 h followed by treatment with 10 ng/ml BMP9 (R&D Systems, 3209-BP-010) at 37°C for 1 h. Immediately following drug treatments, HUVEC were fixed in warm 4% PFA at RT for 4 min. Endothelial cell flow experiments Flow experiments were performed using an Ibidi pump system as previously described (Ruter et al., 2021) with adjustments as follows: HUVEC 48 h post-transfection were seeded onto fibronectin-coated Ibidi slides (μ-Slide I Luer I 0.6 mm, 80186) in flowmedium (EBM-2with 2% FBS, 1× antibioticantimycotic, and 1% nyastatin) at a density of 2×105 cells/mm2.

Techniques: Cell Culture, Staining

Journal: Oncotarget

Article Title: Inhibition of chemerin/CMKLR1 axis in neuroblastoma cells reduces clonogenicity and cell viability in vitro and impairs tumor growth in vivo

doi: 10.18632/oncotarget.19619

Figure Lengend Snippet: Cell viability was determined after 72h of incubation with α-NETA (0.313-10μM). A dose-dependent decrease in cell viability was observed in the neuroblastoma cell lines but not in MRC-5 and HUVEC cells (A) . Data is presented as mean ± SEM from three experiments. The IC 50 values are given with 95% confidence intervals in (B) . The mean of log IC 50 s in neuroblastoma cell lines was significantly lower than the hypothetical log IC 50 of the investigated normal cell lines (one sample t-test, p=0.029; means: 0.726 vs 1). The CMKLR1 antagonist α-NETA reduces the clonogenicity of SK-N-BE(2) cells ( C ; 1.25 and 2.5μM, n=3) and other neuroblastoma cell lines ( D ; 0.313-2.5μM, n=3) in a dose-dependent manner after 72h treatment. Data is presented as mean ± SD from a representative experiment. The experiment was repeated twice more with similar results. Statistical testing was performed using a two-way ANOVA P<0.001 for both stimulation and between cell lines followed by Dunnett's post-test control vs. treatment * P<0.05, *** P<0.001.

Article Snippet: The human fibroblast cell line MRC-5 and human umbilical vein endothelial cells (HUVEC) were purchased from the ATCC and cultivated in EGM-2 BulletKit with 2% FBS (Lonza, Basel, Switzerland) and MEM supplemented with 2mM L-glutamine, 1% non-essential amino acids and 10% FBS, respectively.

Techniques: Incubation, Control

Journal: Scientific Reports

Article Title: Cell Spheroids with Enhanced Aggressiveness to Mimic Human Liver Cancer In Vitro and In Vivo

doi: 10.1038/s41598-017-10828-7

Figure Lengend Snippet: HUVECs promote proliferation and invasion by activation of MAPK pathway via cytokine-mediated signalling in vitro . ( a ) Distribution of 2% of HUVECs in the spheroid was analysed using ICC images of middle and bottom sections of cell spheroids co-cultured with 2% of GFP-tagged HUVECs. Scale bars, 200 μm. ( b ) ELISA of VEGF secretion in culture supernatant in spheroids co-cultured with the indicated concentrations of HUVECs as compared with Huh7 spheroids cultured alone, at 120 h. ( c ) Human cytokine array analysis of Huh7 spheroids cultured alone, with 2% HUVECs, and with 20% HUVECs. ( d ) Huh7-3D or Huh7/2% HUVEC-3D were transferred as single spheroids into each well of 96-well plate and treated with serum-free DMEM containing VEGF-neutralizing antibody (5, 10, 30 µg/ml) for 72 h. Spheroid areas were quantitated using ImageJ software before adding VEGF antibody (0 h) and after 72 h. Error bars display the standard deviation of five independent measurements. ( e ) At 72 h after VEGF antibody treatment, spheroids of each experimental group were harvested and analysed by western blotting with the indicated antibodies. ( f ) Huh7-3D or Huh7/2% HUVEC-3D were embedded into Matrigel in a 24-well plate and treated with serum-free DMEM containing 30 µg/ml VEGF-neutralizing antibody for 5 days. Mouse IgG isotype control antibody (30 µg/ml) was used as negative control. Representative images of each group are shown. ( g ) The sprouting area per experimental group was quantitated with ImageJ. Error bars display the standard deviation of six independent measurements. * P < 0.05; *** P < 0.001.

Article Snippet: HUVECs were purchased from Lonza (Basel, Switzerland) and GFP-tagged HUVECs were purchased from Angio-Proteomie (Boston, MA, USA).

Techniques: Activation Assay, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay, Software, Standard Deviation, Western Blot, Negative Control

Journal: BMC Oral Health

Article Title: Antimicrobial peptides derived from human ameloblastin targeting biofilms

doi: 10.1186/s12903-025-07433-w

Figure Lengend Snippet: AMPs toxicity analysis. ( A ) Haemolytic activity of A, Am, B, Bm measured in the range of 12.5 to 100 (300) µM on a human blood sample and expressed as HC 50 . ( B ) Cytotoxic activity of A, Am, B, Bm peptides measured in the range of 12.5 to 100 (300) µM on HCT 116 and HUVEC cell lines, expressed as IC 50

Article Snippet: Two types of cell cultures were employed for the cytotoxicity assay: primary human umbilical vein endothelial cells (HUVEC) from pooled donors (Lonza, Basel, Switzerland) and human colon carcinoma cell line HCT 116 (ATCC).

Techniques: Activity Assay