Journal: bioRxiv

Article Title: Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

doi: 10.1101/2025.11.07.687176

Figure Lengend Snippet: A-B, Volcano plot (A) and GO term enrichment analysis (STRING) (B) of the proteins physically associated with miP-PSTPIP2 (versus rabbit IgGs; CTL) in human endothelial cells treated with IL-1β; n=3 independent cell batches. The dashed lines mark the significance threshold (P value: 0.05). C, Representative confocal images showing miP-PSTPIP2 and F-actin in human endothelial cells. Nuclei were stained with DAPI. Scale bar: 10 µm D, Co-immunoprecipitation (IP) of miP-PSTPIP2 and β-actin or DAAM1 from human endothelial cell lysates. Similar results were obtained using 2 additional cell batches. E-F, Representative confocal images of the interaction (proximity ligation assay: PLA) between miP-PSTPIP2 and caveoilin-1 (CAV1) (E) or Clathrin Heavy Chain (CHC) (F) in human endothelial cells; Scale bar: 10 µm. Similar results were obtained in 3 additional cell batches. G, Co-immunoprecipitation (IP) of miP-PSTPIP2 and CAV1 or CHC from human endothelial cell lysates. Similar results were obtained using 3 additional cell batches. H, Representative confocal images of AP2A1 and miP-PSTPIP2 in human endothelial cells; Scale bar: 50 µm. Similar results were obtained in 3 additional cell batches.

Article Snippet: Human endothelial cells (passage 4, 90% confluent) were starved of serum in endothelial cell basal medium (EBM, PromoCell, Heidelberg, Germany) containing 0.1% BSA and transduced overnight with adenoviruses to express the FLAG-miP-PSTPIP2 or EGFP as control (250 MOI).

Techniques: Staining, Immunoprecipitation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

doi: 10.1101/2025.11.07.687176

Figure Lengend Snippet: A-B, Representative confocal images and quantification of transferrin uptake by human endothelial cells (A) or porcine aortic endothelial cells (B) overexpressing miP-PSTPIP2 (miP) or EGFP as control (CTL) after incubation with transferrin for the indicated time; n=4-5 independent cell batches (A: two-way ANOVA and Holm-Šídák’s multiple comparisons test); Scale bar: 50 µm. C, Transcytosis of transferrin in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP as control (CTL); n=4 independent cell batches. D-E, Representative confocal images and quantification of Dil-LDL uptake by human endothelial cells (D) or porcine aortic endothelial cells (E) overexpressing miP-PSTPIP2 (miP) or EGFP as control (CTL) after incubation with Dil-LDL for 60 minutes; n=5-7 independent cell batches; Scale bar: 50 µm. F, Transcytosis of Dil-LDL as in C; n=7 independent cell batches. G-H, Uptake (60 minutes) of oxidized LDL (G) or acetylated LDL (H) by human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP as control (CTL); n=4 independent cell batches. All statistical analyses were performed using unpaired Student’s t-tests unless otherwise specified.

Article Snippet: Human endothelial cells (passage 4, 90% confluent) were starved of serum in endothelial cell basal medium (EBM, PromoCell, Heidelberg, Germany) containing 0.1% BSA and transduced overnight with adenoviruses to express the FLAG-miP-PSTPIP2 or EGFP as control (250 MOI).

Techniques: Control, Incubation

Journal: bioRxiv

Article Title: Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

doi: 10.1101/2025.11.07.687176

Figure Lengend Snippet: A, Western blot analysis and quantification of clathrin heavy chain (CHC), low-density lipoprotein receptor (LDLR), Caveolin-1 (CAV1), AP-2 complex subunit alpha-1 (AP2A1), phospho (p-AP2M1) and total AP-2 complex subunit mu (AP2M1) expression in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP as control (CTL). β-Actin or GAPDH were used as loading controls; n=4 independent cell batches. C, Flow cytometry analysis of surface LDLR expression in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP (CTL). Data are shown as mean fluorescence intensity (MFI); n=4 independent cell batches. D, Representative Total Internal Reflection Fluorescence (TIRF) microscopy images of miP-PSTPIP2 and transferrin in human endothelial cells starved of serum overnight and subsequently incubated with AF555–transferrin for 1, 5 or 15 minutes. n=3 independent experiments; scale bar: 20 µm. All statistical analyses were performed using unpaired Student’s t-tests.

Article Snippet: Human endothelial cells (passage 4, 90% confluent) were starved of serum in endothelial cell basal medium (EBM, PromoCell, Heidelberg, Germany) containing 0.1% BSA and transduced overnight with adenoviruses to express the FLAG-miP-PSTPIP2 or EGFP as control (250 MOI).

Techniques: Western Blot, Expressing, Control, Flow Cytometry, Fluorescence, Microscopy, Incubation

Journal: bioRxiv

Article Title: Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

doi: 10.1101/2025.11.07.687176

Figure Lengend Snippet: A, Representative confocal images showing miP-PSTPIP2 and F-actin in human endothelial cells under basal conditions (solvent) and 0, 5, 10, 30 or 60 minutes after cytochalasin D treatment (0.5 µmol/L). Similar results were obtained in 2 additional cell batches. Scale bar: 50 µm . B, Representative confocal images and quantification of F-actin in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP (CTL) treated with solvent or cytochalasin D (0.5 µmol/L, 20 minutes) for 0, 30 and 180 minutes. The quantification shows the extent of endothelial monolayer disruption measured as counts of low intensity pixels in F-actin immunofluorescence images. n=5 independent cell batches (two-way ANOVA and Holm-Šídák’s multiple comparisons test). Scale bar: 50 µm. C, Representative confocal images of the interaction (proximity ligation assay: PLA) between miP-PSTPIP2 and Arp3. Scale bar: 10 µm. Similar results were obtained in 2 additional cell batches. D, Western blot analysis and quantification of Actin-related protein 2 (Arp2), Actin-related protein 3 (Arp3), Vasodilator-stimulated phosphoprotein (VASP), WASP-family verprolin homologous protein 1 (WAVE1), Ras homolog family member A (RhoA) and Cortactin expression in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP as control (CTL). β-Actin or GAPDH were used as loading controls; n=4 independent cell batches (unpaired Student’s t-test). E, Representative confocal images and quantification of mean fluorescence intensity (MFI) per cell of DiI-LDL uptake in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP (CTL), treated with either control inhibitor CK689 or Arp2/3 inhibitor CK666. n=4 independent cell batches (two-way ANOVA and Holm-Šídák’s multiple comparisons test). Scale bar: 50 µm.

Article Snippet: Human endothelial cells (passage 4, 90% confluent) were starved of serum in endothelial cell basal medium (EBM, PromoCell, Heidelberg, Germany) containing 0.1% BSA and transduced overnight with adenoviruses to express the FLAG-miP-PSTPIP2 or EGFP as control (250 MOI).

Techniques: Solvent, Disruption, Immunofluorescence, Proximity Ligation Assay, Western Blot, Expressing, Control, Fluorescence

Journal: bioRxiv

Article Title: Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

doi: 10.1101/2025.11.07.687176

Figure Lengend Snippet: A, Representative images and quantification of human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP (CTL) at 0 h and 48 h after scratching; wound edges are highlighted in blue. Scale bar: 500 µm; n=4 independent cell batches. B, Migration of human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP (CTL) toward 10% FBS. Cell trajectories were recorded over 20 hours. Representative migration tracks from one experimental batch are shown; n=4 independent cell batches. C, Permeability of monolayers of human endothelial cell overexpressing miP-PSTPIP2 (miP) or EGFP (CTL) to fluorescently-labelled 40 kDa dextran. Mean fluorescence intensity (MFI) in the lower chamber of the transwell was measured after incubation for 15 minutes; n=4 independent cell batches. D, Monocyte chemotactic protein-1-induced migration of monocytes through human endothelial cell monolayers overexpressing miP-PSTPIP2 (miP) or EGFP (CTL); n=4 independent cell batches (two-way ANOVA and Holm-Šídák’s multiple comparisons test). All other statistical analyses were performed using unpaired Student’s t-tests.

Article Snippet: Human endothelial cells (passage 4, 90% confluent) were starved of serum in endothelial cell basal medium (EBM, PromoCell, Heidelberg, Germany) containing 0.1% BSA and transduced overnight with adenoviruses to express the FLAG-miP-PSTPIP2 or EGFP as control (250 MOI).

Techniques: Migration, Permeability, Fluorescence, Incubation

Journal: Viruses

Article Title: Impact of SARS-CoV-2 Wuhan and Omicron Variant Proteins on Type I Interferon Response

doi: 10.3390/v17040569

Figure Lengend Snippet: Mechanisms of endothelial dysfunction in COVID-19. The figure illustrates the key mechanisms driving endothelial dysfunction in COVID-19 and their downstream effects on the vascular system that can lead to fatal organ damage. ( A ) Direct SARS-CoV-2 infection: The viral spike protein binds to ACE2 receptors expressed on endothelial cells, facilitating viral entry and replication, leading to cellular damage [ , , ]. ( B ) Systemic inflammation: Elevated pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α, activate endothelial cells, inducing an amplified inflammatory response [ , , , ]. ( C ) Hypercoagulable state: Endothelial injury promotes thrombin generation and platelet aggregation, resulting in the formation of thrombi and widespread vascular occlusion [ , ]. ( D ) Hypoxia: Reduced oxygen delivery due to severe respiratory distress exacerbates endothelial dysfunction, further impairing tissue oxygenation [ , ]. ( E ) Complement activation: Overactivation of the complement cascade causes endothelial damage and contributes to pro-thrombotic states through the generation of C3 and C5 convertases [ , , ].

Article Snippet: They were cultured in Human Large Vessel Endothelial Cell Basal Medium (formerly Medium 200) (Thermo Fisher Scientific) supplemented with Large Vessel Endothelial Supplement (LVES) (Thermo Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), and 100 U/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific).

Techniques: Infection, Amplification, Activation Assay

Journal: F1000Research

Article Title: A humanised thrombus-on-a-chip model utilising tissue-engineered arterial constructs: A method to reduce and replace mice used in thrombosis and haemostasis research.

doi: 10.12688/f1000research.158910.1

Figure Lengend Snippet:

Article Snippet: Human Large Vessel Endothelial Cell Basal Medium (previously known as Medium 200) , Fisher Scientific (Loughborough, UK) , Media for HUVEC culture Must be kept sterile.

Techniques: Sterility, Concentration Assay, Infection, Activity Assay