Journal: The EMBO Journal

Article Title: NPC1 regulates the distribution of phosphatidylinositol 4‐kinases at Golgi and lysosomal membranes

doi: 10.15252/embj.2020105990

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Article Snippet: Protein samples were separated on 4 to 12% polyacrylamide Bis‐Tris gel for 1h15 at 155V and electrophoretically blotted onto polyvinylidene difluoride membranes using a Mini‐Bolt system (A25977; Thermo Fisher Scientific).

Techniques: Recombinant, Electron Microscopy, Blocking Assay, Protease Inhibitor, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Control, Sequencing, Software, Imaging, Membrane

Journal: Scientific Reports

Article Title: Structural and functional characterization of endothelial microparticles released by cigarette smoke

doi: 10.1038/srep31596

Figure Lengend Snippet: ( A ) Abundance of circulating endothelial microparticles (EMPs) in human plasma (demographics in ). Data represent log-transformed CD31+/CD42b- events. ANOVA (p < 0.05; Bartlett’s *p < 0.0001). ( B ) Abundance of EMPs released from primary human lung microvascular (HLMVEC) and human pulmonary artery (HPAEC) endothelial cells exposed to CS or ambient air (AC) extract (v:v %; 2 h). Mean + SEM; ANOVA (p < 0.001; Tukey’s *p < 0.05, n = 5). ( C – E ) Representative images (n = 3) from videos (in ) of HLMVEC transduced with plasma membrane-RFP-BacMan and exposed to AC (( C) protruding lamellipodia: arrowhead; insert); or to CS (5%, 1 h, ( D) and insets; or 2%, 30 min, ( E) ). Note EMPs (yellow arrow) released from the plasma membrane at tips of retracting filopodia (arrowhead). ( F) . Transmission electron microscopy micrograph of heterogeneous human plasma cMPs. ( G ). Abundance and size distribution of EMPs released from AC- and CS-exposed HLMVEC (5%; 2 h) measured by NanoSight-NS300 (Mean+/−SD; n = 3). CS released mostly EMPs 50 nm–200 nm (mode 129 nm; exosome fraction). t-test *p < 0.05 for sizes 82–92 nm, 107–127 nm, 262–277 nm, 372–387 nm, and 790–820 nm.

Article Snippet: Primary HLMVEC and HPAEC were from Lonza (Walkersville, MD).

Techniques: Transformation Assay, Transduction, Transmission Assay, Electron Microscopy

Journal: Biomedicines

Article Title: Essential Role of Rho-Associated Kinase in ABO Immune Complex-Mediated Endothelial Barrier Disruption

doi: 10.3390/biomedicines9121851

Figure Lengend Snippet: Percent change in TEER of HPAEC over 8 h after exposure to matched and mismatched concentrated ABO plasma as compared to media and thrombin. ABO-matched vs unmatched plasma were also compared statistically. Data shown in mean ± SD.

Article Snippet: Human pulmonary artery endothelial cells (HPAEC) of known ABO types were obtained from Lonza (Walkersville, MD, USA) and cultured in gelatin-coated flasks as described [ ].

Techniques:

Journal: Biomedicines

Article Title: Essential Role of Rho-Associated Kinase in ABO Immune Complex-Mediated Endothelial Barrier Disruption

doi: 10.3390/biomedicines9121851

Figure Lengend Snippet: ABO mismatched plasma and IC cause endothelial barrier disruption. An illustration of type A HPAEC; EC were plated on gold electrode arrays and grown to confluency. Cells were treated with ABO identical and non-identical plasma as well as varying concentrations of IC, and transendothelial resistance (TEER) was measured over 3 h by Electric Cell-substrate Impedance Sensing System (ECIS). Resistance was normalized to the values before treatment. Dotted red bars and arrow indicate when measurement was paused and treatments were added.

Article Snippet: Human pulmonary artery endothelial cells (HPAEC) of known ABO types were obtained from Lonza (Walkersville, MD, USA) and cultured in gelatin-coated flasks as described [ ].

Techniques: Electric Cell-substrate Impedance Sensing

Journal: Biomedicines

Article Title: Essential Role of Rho-Associated Kinase in ABO Immune Complex-Mediated Endothelial Barrier Disruption

doi: 10.3390/biomedicines9121851

Figure Lengend Snippet: HPAEC cell–cell adhesion damaged by IC treatment. Type O HPAEC visualized by Scanning Electron Microscopy (SEM) after injury. ( A ) Cells were treated with non-concentrated or concentrated plasma-derived IC at 1:1 concentration with media for the indicated time points and imaged at different magnifications. Scale bar: 10,000×: 1 μm; 5000×: 2 μm; 1500×: 7 μm; 500×: 20 μm. ( B ) Cells were treated with type O plasma for 120 min and imaged at 10,000× as a negative control for the effects of ABO identical plasma. Arrows denote cell–cell contact sites.

Article Snippet: Human pulmonary artery endothelial cells (HPAEC) of known ABO types were obtained from Lonza (Walkersville, MD, USA) and cultured in gelatin-coated flasks as described [ ].

Techniques: Electron Microscopy, Derivative Assay, Concentration Assay, Negative Control

Journal: Biomedicines

Article Title: Essential Role of Rho-Associated Kinase in ABO Immune Complex-Mediated Endothelial Barrier Disruption

doi: 10.3390/biomedicines9121851

Figure Lengend Snippet: ABO mismatched serum and IC decrease cell surface VE-cadherin and induce actin stress fiber formation in HPAEC. Type O HPAEC were plated on coverslips and treated with ( A , B ) concentrated O serum, concentrated A serum, ( C – E ) serum-derived IC 1:1 with media, or ( F – H ) plasma-derived IC 1:25 or 1:1 with media for the indicated time points. Cells were stained with anti-VE-cadherin antibody (green) to mark adherens functions (AJs), Phalloidin-594 (red) to stain actin stress fibers, and DAPI (blue) to label nuclei. ( B , E , G , H ) Mean fluorescence intensity of actin stress fibers (phalloidin staining) per cell was determined. Values are reported in arbitrary units (AU) and analyzed by ANOVA. p -values as follows: ** p < 0.005; **** p < 0.0001; ns = not significant.

Article Snippet: Human pulmonary artery endothelial cells (HPAEC) of known ABO types were obtained from Lonza (Walkersville, MD, USA) and cultured in gelatin-coated flasks as described [ ].

Techniques: Derivative Assay, Staining, Fluorescence

Journal: Biomedicines

Article Title: Essential Role of Rho-Associated Kinase in ABO Immune Complex-Mediated Endothelial Barrier Disruption

doi: 10.3390/biomedicines9121851

Figure Lengend Snippet: Endothelial barrier disruption by IC was prevented by treatment with ROCK inhibitor Y-27632. Type O HPAEC were pre-treated with 10 µM ROCK inhibitor (Y-27632) before injury with concentrated serum-derived IC 1:1 with media. ( A ) Cells were stained with anti-VE-Cadherin antibody (green) to mark AJs, Phalloidin-594 (red) to visualize actin stress fibers, and DAPI (blue) for nuclei. ( B ) Mean fluorescence intensity of actin stress fibers (phalloidin staining) per cell was determined. Values are reported in arbitrary units (AU) and analyzed by two-way ANOVA. The 120 min time point was excluded from statistical calculations due to low cell number in the Control group. ( C ) Gap formation between cells was measured by calculating the area without cell coverage in each field normalized to the total area of the field. ( D ) The number of cells per field was quantitated and averaged for each treatment. Data are mean + S.E. (n = 3 fields per condition) and were analyzed by two-way ANOVA. p -values as follows: ** p < 0.01; **** p < 0.0001.

Article Snippet: Human pulmonary artery endothelial cells (HPAEC) of known ABO types were obtained from Lonza (Walkersville, MD, USA) and cultured in gelatin-coated flasks as described [ ].

Techniques: Derivative Assay, Staining, Fluorescence