Journal: bioRxiv

Article Title: Galectin-1 Modulates Cell Adhesions, Caveolae, and Vascular Permeability in Kidney Endothelial Cells – Insights from Proteomics, Phosphoproteomics, and Functional Studies

doi: 10.64898/2026.03.03.709385

Figure Lengend Snippet: A) Feature plot showing LGALS1 gene expression in immune cells and endothelial cells (purple) and no expression in other cell types (grey), in the healthy human kidney, based on single-cell RNA-Sequencing data from 19 living donor biopsies ( McEvoy et al , 2022 ). B) Expression of galectin-1 in GMECs vs PBMCs by flow cytometry. Galectin-1 Absent Control (orange), PBMC (blue), GMEC (red). C) Galectin-1 protein expression in the cytoplasm and D) on the membrane of primary GMECs stained for galectin-1 (green), DAPI (blue) and actin (red). E) Imaging mass cytometry comparing CD31 and galectin-1 co-expression to F) CD45 and galectin-1 co-expression in a representative kidney biopsy from a patient with ABMR. White boxes magnify the glomerulus. CD31 or CD45 (green), nuclei (blue) and galectin-1 (red). NK cells, Natural Killer cells; GMEC, Glomerular Microvascular Endothelial Cells; FMO, Fluorescence minus one; PBMC, Peripheral Blood Mononuclear Cell; ABMR, Antibody Mediated Rejection.

Article Snippet: Commercial primary human GMECs were purchased from Cell Systems and cultured in Endothelial Cell Growth Media MV (C-22120; Promocell), supplemented with the ready-to-use kit containing 10% (v/v) dialyzed fetal calf serum (FCS), 10 ng/mL epidermal growth factor (EGF), 1 μg/mL hydrocortisone, and 90 μg/mL heparin.

Techniques: Gene Expression, Expressing, Single Cell, RNA Sequencing, Flow Cytometry, Control, Membrane, Staining, Imaging, Mass Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Galectin-1 Modulates Cell Adhesions, Caveolae, and Vascular Permeability in Kidney Endothelial Cells – Insights from Proteomics, Phosphoproteomics, and Functional Studies

doi: 10.64898/2026.03.03.709385

Figure Lengend Snippet: Overview of proteomics/phosphoproteomics workflow, including genetic inhibition of LGALS1 in glomerular microvascular endothelial cells (GMECs), treatment of GMECs with 50U/mL of interferon-γ (IFNγ) or vehicle, sample preparation and phosphopeptide enrichment, followed by LC-MS/MS, data analysis by MaxQuant, statistical analysis to identify differentially expressed proteins by Perseus for total proteomics, or MSStatsPTM for phosphoproteomics, pathway enrichment analysis (PathDIP) ( Pastrello et al , 2024 ), protein-protein interaction analysis (IID) ( Kotlyar et al , 2026 ), and Gene Ontology enrichment analysis ( Ashburner et al , 2000 ; Thomas et al , 2022 ; ), followed by functional assessment in AngioPlate glomerular capillary in vitro . siRNA, small interfering ribonucleic acid; LGALS1 , galectin-1 gene; IFNγ, interferon-γ; LC-MS/MS, liquid chromatography followed by tandem mass spectrometry; PTM, post-translational modification.

Article Snippet: Commercial primary human GMECs were purchased from Cell Systems and cultured in Endothelial Cell Growth Media MV (C-22120; Promocell), supplemented with the ready-to-use kit containing 10% (v/v) dialyzed fetal calf serum (FCS), 10 ng/mL epidermal growth factor (EGF), 1 μg/mL hydrocortisone, and 90 μg/mL heparin.

Techniques: Phospho-proteomics, Inhibition, Sample Prep, Liquid Chromatography with Mass Spectroscopy, Functional Assay, In Vitro, Liquid Chromatography, Mass Spectrometry, Modification

Journal: bioRxiv

Article Title: Galectin-1 Modulates Cell Adhesions, Caveolae, and Vascular Permeability in Kidney Endothelial Cells – Insights from Proteomics, Phosphoproteomics, and Functional Studies

doi: 10.64898/2026.03.03.709385

Figure Lengend Snippet: A) Overview of GMEC treatment and proteome analysis workflow. B) Galectin-1 protein expression was significantly decreased with LGALS1 siRNA treatment. Data are presented as mean ± SEM. Student’s T-test, **P<0.01. C) Proteins identified, quantified, and significant for each treatment and their interaction. Red box denotes proteins significant with LGALS1 siRNA alone. D) Heatmap of 50 proteins included in cluster 3 (complete heatmap is in Supplemental Figure S6 ). Red colour indicates increased expression and blue colour indicates decreased expression, with asterisk denoting galectin-1. E) Bubble plot of top 15 pathways enriched among cluster 3 proteins (Benjamini Hochberg, FDR<0.01). The size of the circle indicates the number of proteins in the pathway, and the colour indicates significance. Pathways extracted from PathDIP ( Pastrello et al , 2024 ). F) Heatmap of 80 proteins included in cluster 1 (complete heatmap is in Supplemental Figure S7 ). Red asterisk denotes proteins mapped to cytoskeleton and focal adhesion pathways. G) Bubble plot of top 15 pathways enriched among cluster 1 proteins (Benjamini Hochberg, FDR<0.01). ECM and focal adhesion pathways are denoted by red asterisk. Pathways extracted from PathDIP ( Pastrello et al , 2024 ) LGALS1 , galectin-1 gene; GMEC, glomerular microvascular endothelial cells; NT, non-targeting control; Bio-rep, biological replicate; ECM, extracellular matrix.

Article Snippet: Commercial primary human GMECs were purchased from Cell Systems and cultured in Endothelial Cell Growth Media MV (C-22120; Promocell), supplemented with the ready-to-use kit containing 10% (v/v) dialyzed fetal calf serum (FCS), 10 ng/mL epidermal growth factor (EGF), 1 μg/mL hydrocortisone, and 90 μg/mL heparin.

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Galectin-1 Modulates Cell Adhesions, Caveolae, and Vascular Permeability in Kidney Endothelial Cells – Insights from Proteomics, Phosphoproteomics, and Functional Studies

doi: 10.64898/2026.03.03.709385

Figure Lengend Snippet: A) Proteins identified, quantified, and significant for each treatment and their interaction; red box denotes proteins significant for the interaction between IFNγ treatment and LGALS1 siRNA. B-D) Bubble plots of top 20 Gene Ontology (GO) terms (Biological Process, Cellular Component, and Molecular Function) enriched among the 267 interaction-significant proteins (FDR: BH<0.01). The size of the circle indicates the number of proteins in the pathway, and the colour indicates significance. Cytoskeleton and focal adhesion terms are denoted by red boxes. E) Protein-protein interaction network (generated using IID ( Kotlyar et al , 2026 )) of significant proteins mapped to focal adhesion, regulation of cytoskeleton organization, anchoring junction, and cell adhesion molecule binding GO terms. The connection between the nodes and mapped GO terms are shown in grey. Physical protein interactions annotated with kidney expression are shown in red. Nodes represented with a blue dot are significant for the interaction between LGALS1 siRNA and IFNγ. Nodes with an outside circle are significant for IFNγ vs vehicle treatment. Nodes with protein names in blue are significant for LGALS1 siRNA vs NT control. F) Nine proteins were shared between the 48 significant interaction proteins mapped to GO terms and the 236 proteins significantly altered by LGALS1 siRNA. The two proteins, ITGB5 (Integrin subunit beta 5) and PLAU (Urokinase-type plasminogen activator), were shared by all 3 datasets, including the 2 previously mentioned and the 8 proteins significantly altered by LGALS1 siRNA, and mapped to cytoskeleton and focal adhesion pathways from . G) Validation of β5 integrin (ITGB5) expression changes using immunoblotting. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was included as a loading control. LGALS1 , galectin-1 gene; GMEC, glomerular microvascular endothelial cells; IFNγ, interferon-γ; GO, Gene Ontology; NT, non-targeting control.

Article Snippet: Commercial primary human GMECs were purchased from Cell Systems and cultured in Endothelial Cell Growth Media MV (C-22120; Promocell), supplemented with the ready-to-use kit containing 10% (v/v) dialyzed fetal calf serum (FCS), 10 ng/mL epidermal growth factor (EGF), 1 μg/mL hydrocortisone, and 90 μg/mL heparin.

Techniques: Generated, Binding Assay, Expressing, Control, Biomarker Discovery, Western Blot

Journal: bioRxiv

Article Title: Galectin-1 Modulates Cell Adhesions, Caveolae, and Vascular Permeability in Kidney Endothelial Cells – Insights from Proteomics, Phosphoproteomics, and Functional Studies

doi: 10.64898/2026.03.03.709385

Figure Lengend Snippet: A) Overview of treatment and phosphoproteome enrichment workflow. B) Phosphopeptides identified, quantified, and significant for each treatment and their interaction after correcting for matched unmodified peptides using MSStatsPTM in R. C) Volcano plots of phosphopeptide Log2FC of IFNγ treatment, LGALS1 siRNA, and both IFNγ and LGALS1 siRNA, compared to non-targeting control and vehicle plotted against –log10(p-value). Orange colour represents non-significant proteins, while blue colour represents those with p<0.01 (linear mixed-effects model), and green represents those with FDR: BH<0.2. D) Heatmap of 28 phosphopeptides significant in at least 1 comparison from panel C. Red colour indicates increased expression, blue colour indicates decreased expression, and black colour indicates phosphopeptides that were not found in that comparison. Colour on the top bar denotes function (obtained from uniport.org ) of the protein the phosphopeptide belongs to. Black and grey asterisks denote FDR<0.2 and p<0.01, respectively. Immunostaining of CAVN1 and CAV1 when GMECs were treated with LGALS1 siRNA, IFNγ, or both. Green colour indicates CAV1, red colour indicates CAVN1. Red arrows denote co-staining of CAV1 and CAVN1 in the NT_V condition. LGALS1 , galectin-1 gene; GMEC, glomerular microvascular endothelial cells; IFNγ, interferon-γ; NT, non-targeting control; NT_V, non-targeting control and vehicle; PTM, post-translational modification.

Article Snippet: Commercial primary human GMECs were purchased from Cell Systems and cultured in Endothelial Cell Growth Media MV (C-22120; Promocell), supplemented with the ready-to-use kit containing 10% (v/v) dialyzed fetal calf serum (FCS), 10 ng/mL epidermal growth factor (EGF), 1 μg/mL hydrocortisone, and 90 μg/mL heparin.

Techniques: Phospho-proteomics, Control, Comparison, Expressing, Immunostaining, Staining, Modification

Journal: bioRxiv

Article Title: Galectin-1 Modulates Cell Adhesions, Caveolae, and Vascular Permeability in Kidney Endothelial Cells – Insights from Proteomics, Phosphoproteomics, and Functional Studies

doi: 10.64898/2026.03.03.709385

Figure Lengend Snippet: A) Schematic diagram of AngioPlate™ preparation and cell seeding. B) Capillaries containing confluent GMECs in AngioPlate™ form tight barrier to both 4kDa and 65kDa dextran on day 7 after seeding, compared to acellular tubes. Data presented as mean ±SEM, n=16-32 per group, Holm-Sidak, ***P<0.001. C) Immunofluorescence image of microvessel stained with CD31 (green) and DAPI (blue). D) Overview of AngioPlate™ timeline including setup, seeding of GMECs pretreated with NT or LGALS1 siRNA, permeability assays post-IFNγ and r-galectin-1 treatments, and the terminal cytokine measurement in the flowthrough. E) Representative immunofluorescence image of the microvessel comparing expression of galectin-1 (red) in LGALS1 siRNA to non-targeting (NT) control. F) Representative immunofluorescence image of the microvessel comparing expression of VE-cadherin (red) in LGALS1 siRNA or NT control, and IFNγ treatment or vehicle. G) GMEC vessel barrier integrity was assessed by diffusion of 4kDa dextran in µg/hour after IFNγ treatment and/or LGALS1 siRNA treatment. Data presented as mean ±SEM, n=15-16 per group, Aligned Rank Sum Test, ***P<0.001 ****P<0.0001. H) GM-CSF and IL-6 cytokine concentrations in flowthrough from wells treated with LGALS1 siRNA, IFNγ, both, or controls. Data presented as mean ±SEM, n=5-6 per group, Aligned Rank Sum Test, *P<0.05, ***P<0.001, ****P<0.0001. I) GMEC vessel barrier integrity was assessed by diffusion of 4kDa dextran in µg/hour after IFNγ Treatment and/or LGALS1 siRNA treatment, followed by the addition of r-galectin-1 or vehicle. Data presented as mean ±SEM, n=7-8 per group, Aligned Rank Sum Test, *P<0.05, ***P<0.001. J) GM-CSF and IL-6 cytokine concentration in flowthrough from wells treated with LGALS1 siRNA, IFNγ, both, or control, followed by the addition of r-galectin-1 or vehicle. Data presented as mean ±SEM, n = 5-6 per group, Aligned Rank Sum Test, *P<0.05, ***P<0.001, ****P<0.0001. GMECs, glomerular microvascular endothelial cells; FITC-dextran, fluorescein conjugated dextran; TRITC-dextran, Tetramethylrhodamine conjugated dextran; CD31, PECAM1 gene; NT, non-targeting control and vehicle; NT_r-galectin-1, non-targeting control and vehicle and recombinant galectin-1; NT_IFNγ, non-targeting control and IFNγ treatment; NT_IFNγ_r-galectin-1, non-targeting control and IFNγ and recombinant galectin-1; siRNA, LGALS1 siRNA and vehicle; siRNA_r-galectin-1, LGALS1 siRNA and vehicle and recombinant galectin-1; siRNA_IFNγ, LGALS1 siRNA and IFNγ treatment; siRNA_IFNγ_r-galectin-1, LGALS1 siRNA and IFNγ and recombinant galectin-1.

Article Snippet: Commercial primary human GMECs were purchased from Cell Systems and cultured in Endothelial Cell Growth Media MV (C-22120; Promocell), supplemented with the ready-to-use kit containing 10% (v/v) dialyzed fetal calf serum (FCS), 10 ng/mL epidermal growth factor (EGF), 1 μg/mL hydrocortisone, and 90 μg/mL heparin.

Techniques: Immunofluorescence, Staining, Permeability, Expressing, Control, Diffusion-based Assay, Concentration Assay, Recombinant