cell culture huvecs  (PromoCell)

Journal: Science Advances

Article Title: Endothelial CLEC5A drives barrier dysfunction and vascular leakage responsible for lung injury in bacterial pneumonia and sepsis

doi: 10.1126/sciadv.adt7589

Figure Lengend Snippet: ( A ) UMAP plot of monocyte clusters in WT lungs ( n = 3) and CLEC5A −/− lungs ( n = 3) and percentage in immune cells. ( B ) Dot plot showing the expression of adhesion molecules in each subtype of endothelial cells. ( C ) GSEA of the DEGs in the endothelial cells between CLEC5A −/− and WT lungs showing significant enrichment of cell-adhesion related-GO pathways, in which the up-regulated DEGs were mainly enriched. The significance was determined by P value with false discovery rate (FDR) < 0.25. ( D ) Mouse PMVECs were isolated and challenged by LPS (10 μg/ml) for 24 hours. ( E ) Levels of MCP-1 and VCAM-1 in the supernatant by ELISA. ( F ) Relative mRNA expression of MCP-1 and VCAM-1 in PMVECs. ( G ) Overview of in vitro adhesion and trans-endothelial migration assays in mouse PMVECs and HUVECs. ( H ) Mouse PBMCs were labeled by BCECF-AM, and the adherent monocytes to PMVECs were observed under a fluorescence microscopy. Scale bar, 50 μm. Monocyte-endothelial adhesion was quantified as the percentage of fluorescence intensity relative to the WT group. ( I ) Transmigration of mouse PBMCs across the PMVEC monolayer was shown as the percentage of transmigrated cells relative to the WT group by MTT assay. Lentivirus-mediated gene delivery for CLEC5A (CLEC5A oe ) or shRNA targeting CLEC5A (CLEC5A sh ) were carried out in HUVECs. ( J ) Adhesion of THP-1 to HUVECs was shown as fluorescence and quantified as the percentage of fluorescence intensity relative to the control group. Scale bar, 50 μm. ( K ) Migration of THP-1 through the HUVEC monolayer shown as the percentage of transmigrated cells relative to the control group. The statistical significance was determined by an unpaired two-tailed t test (or with Welch’s correction) for comparison between two groups and by one-way ANOVA test for comparisons among four groups (biological replicates, n = 4 per group for each experiment).

Article Snippet: HUVECs were cultured in HUVEC culture medium (iCell-h110-001b#, iCell).

Techniques: Expressing, Isolation, Enzyme-linked Immunosorbent Assay, In Vitro, Migration, Labeling, Fluorescence, Microscopy, Transmigration Assay, MTT Assay, shRNA, Control, Two Tailed Test, Comparison

Journal: Science Advances

Article Title: Endothelial CLEC5A drives barrier dysfunction and vascular leakage responsible for lung injury in bacterial pneumonia and sepsis

doi: 10.1126/sciadv.adt7589

Figure Lengend Snippet: To validate SCARB1, PODXL, RAMP2, and FABP4 as potential targets of CLEC5A in vivo, endothelial-specific gene knockdown/overexpression was carried out under Tie1 by AAV9 through tail vein injection. ( A to D ) Survival rate of CLEC5A −/− mice with endothelial knockdown/overexpression of target gene after CLP (biological replicates, n = 10 per group). The statistical significance between survival curves was determined by P value using the log-rank (Mantel-Cox) test. ( E to G ) H&E staining showing histological changes in lung tissues and the corresponding lung injury score (biological replicates, n = 6 per group). Scale bars, 100 μm. ( H and I ) Pulmonary microvascular albumin leakage represented as μg EB/g lung per minute (biological replicates, n = 6 per group). ( J and K ) Levels of TNF-α, IL-6, and MCP-1 in the BALF (biological replicates, n = 6 per group). ( L and M ) Levels of TNF-α, IL-6, and MCP-1 in the lungs (biological replicates, n = 6 per group). ( N and O ) Quantification of inflammatory cells in the BALF, including leukocyte, neutrophil, and monocyte (biological replicates, n = 6 per group). ( P ) HUVECs were coinfected with LV expressing CLEC5A sh and PODXL sh . The monocyte-endothelial adhesion and trans-endothelial migration assays were carried out under LPS stimulation. ( Q ) Adhesion of THP-1 to HUVECs (biological replicates, n = 4 per group). ( R ) Transmigration of THP-1 across HUVECs (biological replicates, n = 4 per group). The statistical significance was determined by an unpaired two-tailed t test (or with Welch’s correction) for comparison between two groups.

Article Snippet: HUVECs were cultured in HUVEC culture medium (iCell-h110-001b#, iCell).

Techniques: In Vivo, Knockdown, Over Expression, Injection, Staining, Expressing, Migration, Transmigration Assay, Two Tailed Test, Comparison

Journal: CNS Neuroscience & Therapeutics

Article Title: Asparagine Endopeptidase Inhibition Attenuates Tissue Plasminogen Activator‐Induced Brain Hemorrhagic Transformation After Ischemic Stroke

doi: 10.1111/cns.70345

Figure Lengend Snippet: AEP inhibitor 7,8‐DHF protect HUVECs against oxygen–glucose deprivation by inhibiting tPA‐induced elevated LRP‐1, MMP2, and MMP9. HUVECs treated with 0.5 μM 7,8‐DHF for 24 h followed by OGD 4 h and reoxygen‐glucose plus tPA (500 ng/mL) for another 24 h as the methods part described. (A, B) Western blotting to evaluate the expression of ZO‐1, claudin5, occluding, and JAM‐1. (C) The viability of HUVECs at different time points. (D) AEP enzymatic analysis of HUVECs subjected to 24 h tPA treatment following 7,8‐DHF and OGD. (E, F) Western blotting to evaluate the expression of AEP, p‐TrkB, TrkB, LRP‐1, MMP2, and MMP9. (A) n = 4, (C) n = 6, (D) n = 4, (E) n = 4 per group. Data are presented as mean ± SEM, and statistical analysis is performed using one‐way ANOVA test followed by Tukey's multiple comparisons test when P value of Levene test > 0.05 or Welch test followed by Dunnett T3 multiple comparisons test when the P value of Levene test < 0.05. Normality and variance are assessed via Shapiro‐Wilk test and Levene's test, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The HUVECs were cultured in complete culture medium for HUVEC (CM‐0122, Procell Life Science & Technology, China).

Techniques: Western Blot, Expressing

Journal: BMC Cancer

Article Title: Exploration of RNA-binding proteins identified RPS27 as a potential regulator associated with Kaposi’s sarcoma development

doi: 10.1186/s12885-025-13790-0

Figure Lengend Snippet: siRPS27 increased the pro-tumor signatures of HUVECs. A Bar plot showing the expression level of RPS27 mRNA in WT, siNC, and siRPS27 samples. B Western blot results show the expression level of RPS27 protein in three groups. The right panel shows the quantitative result. C Cell cycle results show the percentages of three cellular stages in three groups. The right panel shows the quantitative result. D Bar plot showing the cellular proliferation results in three groups. E Cell migration results show the migrated cell numbers in three groups. The right panel shows the quantitative result. F Cell invasion results show the invaded cell numbers in three groups. The right panel shows the quantitative result. G Tube formation results show the tube length in three groups. The right panel shows the quantitative result. * p -value < 0.05; ** p -value < 0.01; *** p -value < 0.001; Student’s t -test

Article Snippet: After culturing each group of cells in serum-free HUVEC cell-specific culture medium for 48 h, a cell suspension was prepared, and 50 μL of cell suspension (1 × 10 5 cells) was seeded in the plate coated with Matrigel and incubated for another 12 h. Representative photographs were subsequently captured under the microscope (IX53, OLYMPUS, Japan) after co-culture.

Techniques: Expressing, Western Blot, Migration