Journal: Clinical Science (London, England : 1979)

Article Title: The loss of glycoprotein nonmetastatic melanoma protein B (GPNMB) alters endothelial cell permeability, metabolism, and survival during infectious challenge

doi: 10.1042/CS20256682

Figure Lengend Snippet: (A ) Interleukin-8 (IL-8), ( B ) tumor necrosis factor-alpha (TNF-α), ( C ) the tissue inhibitors of metalloproteinases 1 (TIMP1) concentrations, and ( D ) metalloproteinases-2, ( E ) metalloproteinases-3, and ( F ) metalloproteinases-9 activity in the culture medium of human microvascular endothelial cells (HMVECs) treated with scrambled siRNA (SCR) or GPNMB-targeting siRNA (siGPNMB), following stimulation with either vehicle or heat-killed E. coli (HKEC, 3 × 10⁸ cells/ml) for 6 h. Each dot represents an individual sample. data are presented as mean ± SEM. Statistical analysis: Two-way ANOVA, followed by the Tukey post hoc test.

Article Snippet: Human dermal microvascular endothelial cells (HMVEC) were obtained from the American Type Culture Collection (ATCC CRL-4060; Manassas, VA, U.S.A.).

Techniques: Activity Assay

Journal: Clinical Science (London, England : 1979)

Article Title: The loss of glycoprotein nonmetastatic melanoma protein B (GPNMB) alters endothelial cell permeability, metabolism, and survival during infectious challenge

doi: 10.1042/CS20256682

Figure Lengend Snippet: (A ) Transendothelial electrical resistance (TEER) of human microvascular endothelial cells (HMVECs) treated with scrambled siRNA (siControl) or GPNMB-targeting siRNA (siGPNMB). ( B ) TEER measurements following lipopolysaccharide (LPS, 100 ng/ml) exposure, normalized to baseline values prior to stimulation. ( C ) TEER measurements after exposure to heat-killed E. coli (HKEC, 3 × 10⁸ cells/ml), normalized to baseline values. ( D-F ) Protein expression levels of ICAM, Integrin-β, and VE-Cadherin, with α-Tubulin used as a loading control. The number of samples used in each experiment ( n ) is expressed in parentheses or dots. Data are presented as mean ± SEM. Statistical analysis: Two-way ANOVA, followed by the Tukey post hoc test.

Article Snippet: Human dermal microvascular endothelial cells (HMVEC) were obtained from the American Type Culture Collection (ATCC CRL-4060; Manassas, VA, U.S.A.).

Techniques: Expressing, Control

Journal: Clinical Science (London, England : 1979)

Article Title: The loss of glycoprotein nonmetastatic melanoma protein B (GPNMB) alters endothelial cell permeability, metabolism, and survival during infectious challenge

doi: 10.1042/CS20256682

Figure Lengend Snippet: Control HMVEC cells (SCR) and treated with siRNA for GPNMB silencing (siGPNMB) were stimulated with vehicle or heat-killed E. coli (HKEC, 3 × 10 8 cells/ml) for 6 h. ( A ) Cell viability (CCK-8 assay) was assessed 1 h after the end of the entire protocol. ( B ) Proliferation (CCK-8 assay) was evaluated at 0-, 12-, and 24 h post-stimulation; the bar graph inserted in the figures represents the area under the curve (AUC) analysis of proliferation at 18 hours. ( C ) Cell migration was assessed using the Scratch-Wounding Assay at 2-, 6-, 12-, and 18 h post-stimulation; the bar graph inserted in the figures represents the quantification of the endpoint of the cell migration curve. ( D ) Representative images of the scratch-wounding assay at 0 and 18 h. ( E-G ) Phosphorylation levels of ERK, JNK, and p38 protein expression were normalized to their respective total protein levels. ( H ) PCNA protein expression normalized to α-Tubulin as a loading control. The number of samples used in each experiment ( n ) is expressed in parentheses or dots. Results are expressed as mean ± SEM. Statistics: Two-way ANOVA, followed by the Tukey post hoc test.

Article Snippet: Human dermal microvascular endothelial cells (HMVEC) were obtained from the American Type Culture Collection (ATCC CRL-4060; Manassas, VA, U.S.A.).

Techniques: Control, CCK-8 Assay, Migration, Phospho-proteomics, Expressing

Journal: Microbiology Spectrum

Article Title: Transendothelial migration of the Lyme disease spirochete involves spirochete internalization as an intermediate step through a transcellular pathway that involves Cdc42 and Rac1

doi: 10.1128/spectrum.02221-24

Figure Lengend Snippet: Transendothelial migration of B. burgdorferi in Transwell chambers. (A) To study B. burgdorferi transmigration, Transwell chambers were seeded with hMVEC-d or hTERT cells until a tight monolayer was formed (<4% albumin diffusion). The upper chamber was infected with 3 × 10 5 spirochetes; after 20 h of infection, we counted the spirochetes in both the upper and lower chambers (either manually or by flow cytometry) and calculated the percentage of total transmigrated spirochetes (lower chamber). ( B, C ) The graphs show the percentage (mean ± SD) of B. burgdorferi that had transmigrated through human microvascular endothelial cells as determined by counting spirochetes in both the upper and lower chamber by flow cytometry. The data represent the mean of % transmigration ±SD of three independent experiments performed in quadruplicate and analyzed for significance using the Mann–Whitney test. NI denotes the non-infectious strain GCB705 (B31-A + pTM61 gent,gfp, see ), and WT indicates GCB726. ( D ) Evaluation of B. burgdorferi transendothelial migration in hTERT treated with AKB-9785. The complete chamber was treated with 5 µM AKB9785 for the duration of the assay. The upper chamber was infected with 3 × 10 5 spirochetes, and the percentage of total transmigrated spirochete was assessed by counting in a Petroff–Hausser chamber and dark-field microscopy after 20 h of infection. The data represent the mean % transmigration ±SD of three independent experiments performed in quadruplicate; non-parametric ANOVA was performed to compare the control cells with the treated cells using the Kruskal–Wallis post-test (ns = not significant). ( E ) Demonstration of the effectiveness of AKB-9785 to lock intercellular junctions in hTERT cells. Ly 294002 at 40 µM was used to disrupt the monolayer, and AKB-9785 was used to lock intercellular junctions and preserve monolayer integrity, which was assessed using an albumin diffusion assay: 10 μg of 555-Alb was added to the upper chamber at 16 h, and the reading was carried out at the final point (20 h). The graph represents the mean ± SD of three experiments, with 1–4 samples for each experimental condition. Statistics were evaluated with the Kruskal–Wallis test and Dunn’s multiple comparison; P < 0.05 was considered significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant.

Article Snippet: Primary human dermal microvascular endothelial cells (hMVEC-d) were purchased from Lonza (CC-2543), grown in Basal Medium (EBM-2) (CC-3156, Lonza) complete media (with supplements EGMTM-2 SingleQuotsTM Supplements [CC-4176, Lonza]) at 37°C under 5% CO 2 and used before passage five. hTERT-immortalized dermal microvascular endothelial cell, neonatal (CRL4060, ATCC), was used. hTERT was cultured in Vascular Cell Basal Medium (VCBM) (PCS-100-030, ATCC) with the microvascular endothelial cell growth kit-BBE (PCS-110-040, ATCC) +0.5 μg/mL puromycin (P8833, Sigma-Aldrich) according to the manufacturer’s instruction.

Techniques: Migration, Transmigration Assay, Diffusion-based Assay, Infection, Flow Cytometry, MANN-WHITNEY, Microscopy, Control, Comparison