Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: A . Physiological rationale: Ambient PM2.5 exposure is epidemiologically linked to increased ischemic stroke risk. This in vitro model simulates the real-life scenario of pre-existing PM2.5 exposure followed by ischemic stroke and subsequent reperfusion. B . Primary adult male HBMEC were exposed to 5, 15, 75, or 300 μg/m 3 PM 2.5 for 48h in total. To compare with the effects of physiological ischemic-like injury, some plates were exposed to hypoxia (1% O 2 ) and glucose deprived media (HGD) for 3h after the initial 24h incubation. Following HGD or normoxia, cells were reperfused with nutrient-enriched media and incubated with PM 2.5 at normoxic (21% O 2 ) conditions as a reference for resolution of ischemia. Barrier integrity, cell viability, reactive oxygen species (ROS), inflammation and LOX-1 expression was assessed. Figure created in BioRender.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: In Vitro, Incubation, Expressing

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: Adult male HBMEC were exposed to vehicle or PM 2.5 (5, 15, 75, or 300 μg/m 3 ) for 24h and incubated for 3h in normoxia- or hypoxia and glucose deprivation (HGD) followed by 24h reperfusion. A . Live cell count (CyQUANT nuclear stain) decreased when exposed to ≥75 μg/m 3 PM 2.5 compared to vehicle. HGD treatment reduced live cell count compared to normoxia but did not differ between particle treated groups. B . Reactive oxygen species (ROS) signal (DCHF-DA) normalized to live cell count. Relative ROS levels increased dose-dependently with PM 2.5 concentration, with significant increase observed at PM 2.5 ≥75 μg/m 3 , in comparison to normoxia vehicle. ROS levels were uniformly elevated following HGD across all doses in comparison to normoxia vehicle and significantly higher than untreated HBMEC. (n=12 technical replicates for vehicle and 5, n=8 technical replicates for 15, 75 and 300) C . Analysis of crystal violet-stained HBMEC shows a longer maximum cellular length when treated with ≥15 μg/m 3 PM 2.5 . (n=21-37 individual cells) D . Representative images of crystal violet-stained HBMEC visualizing a differentiated morphology in cells treated with higher PM 2.5 concentration, where cells appear more elongated and expanding towards neighbouring cells. Data presented as mean ± SD. Statistical significance assessed through Kruskal-Wallis test within treatment groups (Normoxia/HGD) and Mann-Whitney test between groups with different treatment (300 normoxia/vehicle HGD). *p<0.05. ***p<0.001. ****p<0.0001.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: Incubation, Cell Characterization, CyQUANT Assay, Staining, Concentration Assay, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: Western Blot assessment of adult male HBMEC exposed to vehicle, 5, 15, 75, or 300 μg/m 3 PM 2.5 during normoxia or ischemic-like injury with hypoxia, glucose deprivation and reperfusion (HGD). A . Representative Western Blot image of IL-6 and β-actin band migration. B . Signal quantification of 25kDa IL-6 shows no difference between PM 2.5 exposure or HGD treated group. C . Signal quantification of 17kDa IL-6 shows dose-dependency with higher IL-6 expression from higher PM 2.5 exposure, with significant increase ≥75 μg/m 3 and from HGD treatment compared to vehicle. D . Representative Western Blot image of LOX-1 and β-actin. E . Signal quantification of LOX-1 displays a dose-dependent increase in LOX-1 with exposure to ≥15 μg/m 3 PM 2.5 or HGD. (n=4-7 technical replicates). Data presented as mean +-SD. Statistical significance assessed by Kruskal-Wallis test. *p<0.05, **p<0.01.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: Western Blot, Migration, Expressing

Journal: Journal of Pathology and Translational Medicine

Article Title: The Diagnostic Usefulness of HMGA2, Survivin, CEACAM6, and SFN/14-3-3 δ in Follicular Thyroid Carcinoma

doi: 10.4132/jptm.2015.01.31

Figure Lengend Snippet: Immunohistochemistry in AG, FA, FTC and its association with histologic diagnosis

Article Snippet: Monoclonal antibodies were used for Gal-3 (clone 9C4, Novocastra, Newcastle, United Kingdom), HBME1 (clone HBME-1, Dako, Carpinteria, CA, USA), CK19 (clone RCK108, Dako), cyclin D1 (clone SP4, Thermo Fisher Scientific, Waltham, MA, USA), CEACAM6 (clone 9A6, Abcam, Cambridge, MA, USA) and SFN/14-3-3 δ (clone 5D7, Santacruz, Dalla, TX, USA).

Techniques: Immunohistochemistry

Journal: Journal of Pathology and Translational Medicine

Article Title: The Diagnostic Usefulness of HMGA2, Survivin, CEACAM6, and SFN/14-3-3 δ in Follicular Thyroid Carcinoma

doi: 10.4132/jptm.2015.01.31

Figure Lengend Snippet: Diagnostic values of HBME1/HMGA2 for malignancy (FTC vs FA and AG)

Article Snippet: Monoclonal antibodies were used for Gal-3 (clone 9C4, Novocastra, Newcastle, United Kingdom), HBME1 (clone HBME-1, Dako, Carpinteria, CA, USA), CK19 (clone RCK108, Dako), cyclin D1 (clone SP4, Thermo Fisher Scientific, Waltham, MA, USA), CEACAM6 (clone 9A6, Abcam, Cambridge, MA, USA) and SFN/14-3-3 δ (clone 5D7, Santacruz, Dalla, TX, USA).

Techniques: Diagnostic Assay, Marker

Journal: Journal of Pathology and Translational Medicine

Article Title: The Diagnostic Usefulness of HMGA2, Survivin, CEACAM6, and SFN/14-3-3 δ in Follicular Thyroid Carcinoma

doi: 10.4132/jptm.2015.01.31

Figure Lengend Snippet: Diagnostic values of markers for FN (FTC and FA vs AG)

Article Snippet: Monoclonal antibodies were used for Gal-3 (clone 9C4, Novocastra, Newcastle, United Kingdom), HBME1 (clone HBME-1, Dako, Carpinteria, CA, USA), CK19 (clone RCK108, Dako), cyclin D1 (clone SP4, Thermo Fisher Scientific, Waltham, MA, USA), CEACAM6 (clone 9A6, Abcam, Cambridge, MA, USA) and SFN/14-3-3 δ (clone 5D7, Santacruz, Dalla, TX, USA).

Techniques: Diagnostic Assay, Marker

Journal: Nature Communications

Article Title: Strength of Neisseria meningitidis binding to endothelial cells requires highly-ordered CD147/β 2 -adrenoceptor clusters assembled by alpha-actinin-4

doi: 10.1038/ncomms15764

Figure Lengend Snippet: ( a ) CD147 and the β 2 AR colocalize at endothelial cell junctions and at sites of bacterial adhesion. HBMECs, transiently transfected with β 2 AR-YFP, were non-infected (top) or infected with meningococci for 2 h (bottom). CD147 (red) and β 2 AR-YFP (green) receptor localization was analysed by confocal microscopy (scale bars, 10 μm). ( b ) Constructs used for BRET assays. CD147 fused to Renilla luciferase (CD147-Rluc) and β 2 AR fused to YFP (β 2 AR-YFP) were used as energy donor and acceptor, respectively. ( c ) BRET analysis of CD147–β 2 AR interaction. HEK293 cells were transfected with CD147-Rluc (2 ng) and increasing amounts (0–400 ng) of β 2 AR-YFP or CCR5-YFP. At 24 h after transfection, coelenterazine h (Rluc substrate) was added to measure the BRET signal. Results correspond to one experiment (mean±s.e.m., n =3) representative of five independent experiments. ( d ) Isoproterenol modified the BRET 50 . HEK293 cells were transfected with 2 ng CD147-Rluc and 20 ng β 2 AR-YFP, so that energy transfer was close to the BRET50 value. Coelenterazine h (Rluc substrate) was added to measure the basal energy transfer, then 10 μM isoproterenol alone or in combination with 10 μM propranolol, or solvent alone used as control, were added to cells and the energy transfer was measured every 2 min for 20 min. Arrow indicates time of injection of the different ligands. Results correspond to one experiment (mean±s.e.m., n =3) representative of four independent experiments. ( e ) The meningococcal pilin PilE does not modify the BRET 50 . HEK293 cells were transfected with 2 ng CD147-Rluc and 20 ng β 2 AR-YFP, so that energy transfer was close to the BRET50 value. Coelenterazine h was added to measure the basal energy transfer, then purified meningococcal PilE was added and the transfer was measured every 2 min for 30 min. Results correspond to one experiment (mean±s.e.m., n =3) representative of four independent experiments.

Article Snippet: HBMECs, a human bone marrow capillary endothelial cell line provided by Dr B. Weksler , were cultured and infected as described previously .

Techniques: Transfection, Infection, Confocal Microscopy, Construct, Luciferase, Modification, Solvent, Control, Injection, Purification

Journal: Nature Communications

Article Title: Strength of Neisseria meningitidis binding to endothelial cells requires highly-ordered CD147/β 2 -adrenoceptor clusters assembled by alpha-actinin-4

doi: 10.1038/ncomms15764

Figure Lengend Snippet: ( a ) Actn4 co-immunoprecipitates with CD147. HBMECs were either non-infected (Ni) or infected with meningococci for the indicated period of time before lysis. CD147 was immunoprecipitated (irrelevant antibody (Ig) as control) and the obtained samples were successively immunoblotted with anti-Actn4 (top) and anti-CD147 antibodies (bottom). The total cell lysate (TCL) was loaded as a control. ( b ) CD147 and Actn4 colocalize at endothelial cell junctions and at sites of bacterial adhesion. HBMECs non-infected (top) or infected for 2 h with meningococci (bottom) were stained for CD147 (green), Actn4 (red) and actin (blue) and analysed by confocal microscopy. Merged images of the same fields are presented on the right (scale bar, 10 μm). ( c ) Actn4 accumulates at sites of bacterial adhesion independently of PI(4,5)P 2 production and cortical actin polymerization. HBMECs were transfected with GFP-tagged PH domain of PLC-δ1, along with the membrane-targeted FRB-CFP and the mRFP-FKBP domain constructs (with or without 5′-phosphatase domain as indicated), infected with meningococci for 2 h in the presence of rapamycin to induce the translocation of 5′-phosphatase to the plasma membrane and stained for ezrin, actin, CD147 and Actn4. The colour bar indicates the pixel intensity of normalized fluorescence images (in arbitrary units). Arrows point at sites of bacterial adhesion. Control GFP, mRFP-FKBP, mRFP-FKBP-5-phosphatase and FRB-CFP fluorescence images are shown in . Images are representative of five independent experiments (scale bars, 10 μm).

Article Snippet: HBMECs, a human bone marrow capillary endothelial cell line provided by Dr B. Weksler , were cultured and infected as described previously .

Techniques: Infection, Lysis, Immunoprecipitation, Control, Staining, Confocal Microscopy, Transfection, Membrane, Construct, Translocation Assay, Clinical Proteomics, Fluorescence

Journal: Nature Communications

Article Title: Strength of Neisseria meningitidis binding to endothelial cells requires highly-ordered CD147/β 2 -adrenoceptor clusters assembled by alpha-actinin-4

doi: 10.1038/ncomms15764

Figure Lengend Snippet: ( a and b ) Actn4 expression modulates bacterial adhesion to human endothelial cells under shear stress. HBMECs were transfected with ( a ) control (CTL) or Actn4 siRNA or with ( b ) control (CTL) or Actn4 plasmid. Right panels: Western blot analysis of Actn4 levels, using clathrin as a control. Middle panels: Number of adhesion events (meningococci adhering individually or in aggregates) following a 10 min infection under shear stress (0.04 dynes cm −2 ). Left panels: Average size (in pixels) of the meningococcal colony. The number and the size of the adherent individual bacteria and/or bacterial aggregates were quantified using the ImageJ software. Representative images of the adherent bacteria in each condition are shown in . Mean±s.e.m., n =4, **** P <0.0001, ** P <0.01, * P <0.05, two-tailed Student's t -test. ( c ) The meningococcal pilin PilE and PilV selectively interact with the endothelial cell surface. AFM analysis of retraction forces between cantilever tips functionalized with PEG linker alone (control) or with PEG linker plus MBP-pilins (ComP, PilE or PilV) and the apical membrane of living HBMECs. Bar plots indicate the percentage of force curves with unbinding events. Mean±s.e.m.; n =7 experiments (448 force curves and 7 cells per condition), *** P <0.001, one-way analysis of variance (ANOVA). ( d – g ) Actn4 expression regulates interaction strength between meningococcal pilins and their endothelial receptors. ( d ) Schematic representation of the different parameters measured. The detachment force corresponds to the total rupture force of adhesive bonds involved during interaction of pilin-coated bead with the cell surface throughout a series of unbinding events, as measured by the jumps on the force–distance retraction curve. Detachment work represents the ‘work of deadhesion' that contains both mechanical components inherent to the cell type and adhesive elements due to pilin–receptor complexes that break on retraction of the bead. ( e ) AFM analysis of the interaction between 2-μm-sized beads coated with ComP or PilE and living Actn4-depleted or control HBMECs. Shown are the ( f ) detachment forces and the ( g ) detachment work measured. Mean±s.e.m.; n ≥6 experiments (270 force curves and 30 cells per condition), * P <0.05, ** P <0.01, one-way ANOVA.

Article Snippet: HBMECs, a human bone marrow capillary endothelial cell line provided by Dr B. Weksler , were cultured and infected as described previously .

Techniques: Expressing, Shear, Transfection, Control, Plasmid Preparation, Western Blot, Infection, Bacteria, Software, Two Tailed Test, Membrane, Adhesive