Journal: bioRxiv

Article Title: A mycobacterial Sec61 inhibitor disrupts lysosome function by blocking Vacuolar-ATPase biosynthesis

doi: 10.1101/2025.08.26.671788

Figure Lengend Snippet: A. Cartoon structure of V-ATPase complex, depicting the cytosolic V 1 complex responsible for ATP hydrolysis and the membrane embedded V 0 which pumps protons across the membrane. Image generated using Biorender. B. V-ATPase subunits detected in our HDMEC membrane proteome (PRIDE Project PXD037489)( Hsieh et al ., 2025 ) showing predicted sensitivity to mycolactone based on location and topology combined with the known specificity of mycolactone towards different Sec61 substrates, along with a heat map of Log2 fold-change in V-ATPase subunit expression following mycolactone exposure for 24hr. **; p<0.01. C. Immunoblots from HDMEC incubated with 0.02% DMSO or 10ng/ml mycolactone and HeLa cells incubated with 0.05% DMSO for 24hr or 31.25ng/ml mycolactone for various times and stained with the indicated antibodies. GAPDH is included as a loading control. Approximate molecular weights in kDa are indicated for each target. Images are representative of at least n=2 independent experiments.

Article Snippet: Juvenile, single donor human microvascular endothelial cells (HDMEC) were cultured in hVEGF containing Endothelial cell growth medium 2 (Promocell) at 37°C and 5% CO 2 .

Techniques: Membrane, Generated, Expressing, Western Blot, Incubation, Staining, Control

Journal: Function

Article Title: Molecular and Functional Characterization of the Peritoneal Mesothelium, a Barrier for Solute Transport

doi: 10.1093/function/zqae051

Figure Lengend Snippet: Mesothelial and endothelial cell type specific expression of transport related genes, and of tight junction proteins CLDN1 and CLDN5, (A) Expression map of cell junctions, transmembrane channels and transporters, and of transcytotic carriers in human primary peritoneal mesothelial cells (HPMC), immortalized pleural mesothelial cells (MeT-5A), human umbilical vein endothelial cells (HUVEC) and human primary cardiac microvascular endothelial cells (HCMEC). Sealing tight junction claudin1 (CLDN1) is only expressed in mesothelial cells and CLDN5 only in endothelial cells. (B) Immunocytochemical staining of CLDN1 protein in HPMC and MeT-5A, and CLDN5 in HUVEC and HCMEC, together with anchoring protein ZO-1. Pearson correlation analysis of the green and red channel colocalization and RGB spectra at the cell membrane. Scale bar = 10 µm. (C) Quantification of CLDN1 and -5 relative to ZO-1 immunofluorescence in the four cell lines at the cell membrane area (z-stack spacing 0.25 µm). (D) Representative Western blot analysis of CLDN1 and CLDN5 proteins (total protein extraction).

Article Snippet: Human cardiac microvascular endothelial cells (HCMEC), human umbilical vein endothelial cells (HUVEC), and immortalized mesothelial cell line (MeT-5A) were commercially obtained (HUVEC and HCMEC from PromoCell, Heidelberg, Germany; MeT-5A from LGC Standards, Wesel, Germany).

Techniques: Expressing, Staining, Membrane, Immunofluorescence, Western Blot, Protein Extraction

Journal: Function

Article Title: Molecular and Functional Characterization of the Peritoneal Mesothelium, a Barrier for Solute Transport

doi: 10.1093/function/zqae051

Figure Lengend Snippet: Transepithelial resistance, creatinine transport (0.11 kDa), and 4-, 10-, and 70-kDa dextran permeability of polarized mesothelial and endothelial cell monolayers. (A) TER of the four cell lines with increasing cell monolayer density in Transwells, with stable TER being reached with confluence. Confluent HCMEC have a 2.5-fold lower TER, reflecting higher ionic conductance ( n = 5 experiments, 4-5 replicates, P < 0.0001 for HCMEC versus all). (B) The decline in creatinine concentrations in the apical Transwell compartment relative to the initial concentration (10 mg/dL) is given on the left graph. Volume of the basolateral compartment is five times higher (1 mL). The right graph gives the creatinine appearance relative to the creatinine added to the apical compartment and corrected for the higher basolateral volume. The dashed lines indicate the expected equilibration levels between compartments. Transport of creatinine is highest across confluent HCMEC cell monolayers ( P < 0.0001/0.0001 for changes in the apical and basolateral compartment with 4 cell lines, and P < 0.0001/0.0001 for HPMC versus HCMEC only; two-way repeated measure ANOVA; n = 4 experiments, 3 replicates per experiment). (C) 4, 10 and 70 kDa dextran permeabilities of mesothelial and endothelial cell monolayers were calculated from 2 h and 4 h dextran transport kinetics. Paracellular permeability is higher for the smaller macromolecular dextrans across human microvascular endothelial cells. two-way repeated measures ANOVA P < 0.0001 for dextran size, P < 0.0001 for cell type; n = 4 experiments, 3 replicates per experiment, data area mean ± SD, ** P < 0.01, **** P < 0.0001). HCMEC = human cardiac microvascular endothelial cells, HUVEC = human umbilical vein endothelial cells, HPMC = human peritoneal mesothelial cells, MeT-5A = immortalized pleural mesothelial cells.

Article Snippet: Human cardiac microvascular endothelial cells (HCMEC), human umbilical vein endothelial cells (HUVEC), and immortalized mesothelial cell line (MeT-5A) were commercially obtained (HUVEC and HCMEC from PromoCell, Heidelberg, Germany; MeT-5A from LGC Standards, Wesel, Germany).

Techniques: Permeability, Concentration Assay

Journal: bioRxiv

Article Title: The proton-sensing GPR4 receptor regulates paracellular gap formation and permeability of vascular endothelial cells

doi: 10.1101/601088

Figure Lengend Snippet: Plasma membrane staining and paracellular gap area quantitation of ECs treated for up to 5 hours under physiological or acidic pH. Acidosis increases EC gap formation when compared to physiological pH treatment conditions. (A) Representative pictures of plasma membrane staining in Human Umbilical Vein Endothelial Cells (HUVECs) at 0, 3, and 5 hours treated under physiological or acidic pH. Quantitative analysis of gap formation in (B) HUVECs, (C) Human Pulmonary Artery Endothelial Cells (HPAECs), (D) Human Colon Microvascular Endothelial Cells (HMVEC-Colon), and Human Lung Microvascular Endothelial Cells (HMVEC-Lung) over 5 hours. All experiments were performed in triplicate and are representative of four experiments. Data at each time point are presented as mean ± SEM and analyzed for statistical significance between the pH 7.4 group and the pH 6.4 group using the unpaired t -test where **p<0.01 and ***p<0.001. White arrows point to paracellular gaps. Scale bar = 100µm.

Article Snippet: Primary human umbilical vein endothelial cells (HUVEC), human pulmonary artery endothelial cells (HPAEC), and human lung microvascular endothelial cells (HMVEC-Lung) (Lonza, Walkersville, MD, USA), and primary human colon microvascular endothelial cells (HMVEC-Colon) (Cell Biologics Inc., Chicago, IL, USA) were cultured at 37°C and 5% CO 2 in a humidified incubator for experimentation.

Techniques: Staining, Quantitation Assay

Journal: PLoS Pathogens

Article Title: EphA2 contributes to disruption of the blood-brain barrier in cerebral malaria

doi: 10.1371/journal.ppat.1008261

Figure Lengend Snippet: (A) Ephrin-A1 ligand transcription in HBMECs incubated for 1–24 hours with human TNF-α, LT-β, or P . falciparum 3D7-infected RBC lysates ( Pf pRBC) (n = 3 endothelial preparations/group/time point). Values are normalized to untreated cells. Asterisk indicates p<0.05. (B) Ephrin-A1 ligand transcription in MBMECs incubated for 24 hours with naïve RBC lysates (nRBC), Pb A-infected RBC lysates ( Pb A pRBC), or no RBC lysate (∅ RBC) plus mouse LT-α, TNF-α, or media (M)) (n = 3 endothelial preparations/group). (C) Levels of soluble ephrin-A1 ligand in the plasma of children living in an area in Cameroon endemic for P . falciparum malaria. Patients were categorized by admission to the hospital for neurological complications (n = 51), uncomplicated malaria (n = 50), or uninfected and presenting for routine pediatric tests (n = 49). Each dot represents an individual patient. (D) Levels of soluble ephrin-A1 ligand in the plasma of C57BL/6J mice infected with with Pb A (n = 13) or Pb NK65 (n = 13) at day 6 post-infection compared with naïve mice (n = 10). (E) Ephrin-A1 ligands released from MBMECs derived from EphA2-/- or littermate control mice and incubated for 24 hours with no RBC lysate (∅ RBC), naïve RBC lysate (nRBC), and Pb A-infected RBC lysate ( Pb A pRBC) with the addition of media (M), recombinant mouse LT-α (L), or TNF-α (T) (n = 4 endothelial preparations). (F) Ephrin-A1 ligands in the plasma of EphA2-/- or littermate control mice at day 6 post-infection with Pb A (n = 14-15/group) compared to naïve mice (N) (n = 6/group). Bars in all graphs represent the mean ± SEM. Statistical analyses: Two-way ANOVA (A, E), Kruskal-Wallis and Dunn’s multiple comparisons tests (B, D, F), and General linear modeling and Tukey’s pairwise comparison post-ANOVA (C). Only statistically significant (p<0.05) values are shown. Figures represent combined data from 3 (A, B, D, F), or 4 (E) independent experiments.

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) used in this study were either gifted or obtained from a commercial source (Creative Dynamics Inc).

Techniques: Incubation, Infection, Clinical Proteomics, Derivative Assay, Control, Recombinant, Comparison

Journal: PLoS Pathogens

Article Title: EphA2 contributes to disruption of the blood-brain barrier in cerebral malaria

doi: 10.1371/journal.ppat.1008261

Figure Lengend Snippet: The breakdown of the blood-brain barrier during blood-stage Pb A infection begins with parasitized red blood cells (pRBCs) in the schizont stage traveling through the bloodstream and adhering to various receptors expressed on brain microvascular endothelial cells including EPCR, ICAM-1, and other unknown receptors that have yet to be identified (1) . Signaling through these receptors leads to endothelial activation (2) and release of various pro-inflammatory cytokines and chemokines. The cytokine LT-α can act on proximal endothelial cells to induce upregulation of the receptor EphA2 (3) while TNF-α induces upregulation of ephrin-A1 ligand (4) which can be cleaved by metalloproteinases and released into the bloodstream (although this monomeric form is not believed to signal). Chemokines such as CXCL10 and CCL2 recruit circulating immune cells, including CD8+ T cells, to the brain to the site of inflammation (5) . Upon entry into the brain microvasculature, CD8+ T cells expressing ephrin-A1 ligand bind to EphA2 expressed on brain endothelial cells leading to clustering and activation of EphA2. Forward signaling cascades from the EphA2 receptor lead to activation of the NFκB pathway (6) which results in various downstream consequences including disruption of endothelial cell junctions due to both internalization and shedding of different adherens and tight junction protein components (7) . Once brain endothelial cell junctions are disrupted, contents of the vasculature can leak into the brain parenchyma (8) leading to vascular leakage, brain edema, and the development of other neurological symptoms associated with Pb A infection. In the absence of EphA2 upregulation or activation ( i . e . EphA2 deficiency, PbNK65 infection, therapeutic targeting), this endothelial junction disruption does not occur and the blood-brain barrier remains intact.

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) used in this study were either gifted or obtained from a commercial source (Creative Dynamics Inc).

Techniques: Infection, Activation Assay, Expressing, Disruption

Journal: Cell Reports Medicine

Article Title: Broad auto-reactive IgM responses are common in critically ill patients, including those with COVID-19

doi: 10.1016/j.xcrm.2021.100321

Figure Lengend Snippet: COVID-19 patient plasma contains autoantibodies that bind diverse cell types (A) The presence of auto-Ig was detected in human plasma by flow cytometry. Following initial gating on single and live cells (top row), populations were queried for surface-bound antibodies. Fluorescence minus one (FMO) samples (middle row) and an IgG-positive control were used to determine the IgG + gate (bottom left), while gates for IgA + and IgM + events were informed by FMO samples and strategic gating to restrict positive events below 2% in at least half and below 10% in all healthy donor samples (bottom middle and bottom right, respectively). Representative flow cytometry plots are shown. (B–D) Imaging flow cytometry detected auto-IgM (pseudocolored red) bound to the plasma membrane of a human primary alveolar epithelial cell (HPAEC) stained with patient plasma containing a high level of auto-IgM (B). This was not observed in cells incubated with patient plasma without HPAEC-reactive auto-IgM (C) or with plasma obtained from a healthy human control (D). Nuclei are pseudocolored green. Scale bar, 10 μm. IgM-stained plasma membrane indicated by white arrowheads. Representative images are shown. (E) The maximum observed auto-Ig staining percentage across all cell types, from each patient, are shown. (F) Detected auto-Ig levels in specific cell types are shown, per patient. For (E) and (F), the ICU label designates non-COVID ICU patients; the Hyper-γ or H-γ label indicates samples from patients with hypergammaglobulinemia. Primary cells used were human kidney glomerular endothelial cells (HKGECs), human small airway epithelial cells (HSAECs), human small intestinal microvascular endothelial cells (HSIMECs), and human pulmonary airway epithelial cells (HPAECs). Because of sample constraints, each stain was performed once.

Article Snippet: Human Small Intestine Microvascular Endothelial Cells , Neuromics , HEC15.

Techniques: Flow Cytometry, Fluorescence, Positive Control, Imaging, Staining, Incubation

Journal: Cell Reports Medicine

Article Title: Broad auto-reactive IgM responses are common in critically ill patients, including those with COVID-19

doi: 10.1016/j.xcrm.2021.100321

Figure Lengend Snippet:

Article Snippet: Human Small Intestine Microvascular Endothelial Cells , Neuromics , HEC15.

Techniques: Recombinant, Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Software