Journal: Biomolecules

Article Title: CD45 and Basigin (CD147) Are Functional Ligands for Galectin-8 on Human Leukocytes

doi: 10.3390/biom15091243

Figure Lengend Snippet: Validation of Gal-8 chromatography columns. ( A ) Schematic representation of Gal-8 chromatography with HL-60 cell membrane preparations and steps in the procedure. Gal-8 chromatography (1 mg Gal-8/mL beads) with ( B ), positive control: mouse laminin (LA); ( C ) negative control: CHO-Lec8 cell mutant membrane (250 μg/mL); ( D ) HL-60 cell membrane extract (250 μg/mL). ( B – D ) were visualized with silver nitrate staining. ( E , F ) Blotting of fractions from ( D ) with biotinylated Gal-8 (1 μg/mL) in absence ( E ) and presence ( F ) of lactose. Legend: MW, molecular weight markers; LA, laminin; MB, total membrane fraction; FT, flow through; W, wash; S, 100 mM sucrose wash; L, 100 mM lactose elution.

Article Snippet: After SDS-PAGE and transferring the blot with Transblot (Bio-Rad, Hercules, CA, USA), biotinylated cell surface proteins were detected by Streptavidin-horseradish peroxidase (Strep-HRP, Vector Labs, Newark, CA, USA) at a 1:30,000 dilution in Tris buffer saline (TBS) containing 0.1% Tween-5% bovine serum albumin (TBST-BSA) after blocking overnight at 4 °C or 1 h at room temperature (RT) in TBST-BSA.

Techniques: Biomarker Discovery, Chromatography, Membrane, Positive Control, Negative Control, Mutagenesis, Staining, Molecular Weight

Journal: Biomolecules

Article Title: CD45 and Basigin (CD147) Are Functional Ligands for Galectin-8 on Human Leukocytes

doi: 10.3390/biom15091243

Figure Lengend Snippet: Gal-8 chromatography with intact HL-60 cells. ( A ) Schematic representation of Gal-8 chromatography steps with intact HL-60 cells. ( B ) Gal-8 chromatography (1 mg Gal-8/mL beads) of intact, cell surface biotinylated HL-60 cells. Samples were visualized with silver nitrate staining. ( C ) Strep-HRP blot (1 μg/mL) of fractions from B with intact biotinylated HL-60 cells. Legend: MW, molecular weight markers; MB, total membrane fraction; FT, flow through; W, wash; S, 100 mM sucrose wash; L, 100 mM lactose elution.

Article Snippet: After SDS-PAGE and transferring the blot with Transblot (Bio-Rad, Hercules, CA, USA), biotinylated cell surface proteins were detected by Streptavidin-horseradish peroxidase (Strep-HRP, Vector Labs, Newark, CA, USA) at a 1:30,000 dilution in Tris buffer saline (TBS) containing 0.1% Tween-5% bovine serum albumin (TBST-BSA) after blocking overnight at 4 °C or 1 h at room temperature (RT) in TBST-BSA.

Techniques: Chromatography, Staining, Molecular Weight, Membrane

Journal: Biomolecules

Article Title: CD45 and Basigin (CD147) Are Functional Ligands for Galectin-8 on Human Leukocytes

doi: 10.3390/biom15091243

Figure Lengend Snippet: CD45 was detected among the Gal-8 binding proteins on HL-60 cells. ( A ) Anti-CD45 blot of full-length Gal-8 chromatography with HL-60 cell membranes; ( B ) anti-CD45 blot of Gal-8C chromatography with HL-60 cell membranes; ( C ) Reverse Transcriptase-PCR of CD45 isoforms and beta-actin with HL-60 and Jurkat cells. Specific amplicons for CD45 (about 200, 350, and 500 bp) are visible; ( D ) HL-60 cell staining with anti-CD45-Alexa 488 or isotype control measured by flow cytometry; ( E ) confocal images of HL-60 cells treated at 4 °C with biotinylated Gal-8CM ( top row ) or Gal-8NM ( bottom row ) with Strep-Alexa 633 (red) and anti-CD45-Alexa 488 (green); Merge 1: whole cell reconstruction, Merge 2: detailed colocalization (yellow), scale bar = 5 μm. White arrows indicate colocalization in punctate membrane microdomains. Legend: MB, total membrane fraction; FT, flow through; W, wash; S, 100 mM sucrose wash; L, 100 mM lactose elution; MW, molecular weight.

Article Snippet: After SDS-PAGE and transferring the blot with Transblot (Bio-Rad, Hercules, CA, USA), biotinylated cell surface proteins were detected by Streptavidin-horseradish peroxidase (Strep-HRP, Vector Labs, Newark, CA, USA) at a 1:30,000 dilution in Tris buffer saline (TBS) containing 0.1% Tween-5% bovine serum albumin (TBST-BSA) after blocking overnight at 4 °C or 1 h at room temperature (RT) in TBST-BSA.

Techniques: Binding Assay, Chromatography, Reverse Transcription, Staining, Control, Flow Cytometry, Membrane, Molecular Weight

Journal: Biomolecules

Article Title: CD45 and Basigin (CD147) Are Functional Ligands for Galectin-8 on Human Leukocytes

doi: 10.3390/biom15091243

Figure Lengend Snippet: Both N- and C-terminal domains of Gal-8 bind to basigin and colocalize with basigin on HL-60 cells. ( A ) Anti-basigin (CD147) blot of Gal-8NM and Gal-8CM chromatography with HL-60 cell membranes (U: Unbound, B: Bound); ( B ) HL-60 cell staining with anti-basigin-Alexa 488 or isotype control measured by flow cytometry; ( C ) confocal images of HL-60 cells treated at 4 °C with (a) biotinylated Gal-8NM or (e) Gal-8CM with Strep-Alexa 633 (red) and (b,f) anti-basigin-Alexa 488 (green); Merged images Gal-8NM/anti-basigin and Gal-8CM/anti-basigin are shown (c,g), and zoomed images on two individual cells from (c,g) are shown (d,h), respectively. Scale bar represents 5 μm.

Article Snippet: After SDS-PAGE and transferring the blot with Transblot (Bio-Rad, Hercules, CA, USA), biotinylated cell surface proteins were detected by Streptavidin-horseradish peroxidase (Strep-HRP, Vector Labs, Newark, CA, USA) at a 1:30,000 dilution in Tris buffer saline (TBS) containing 0.1% Tween-5% bovine serum albumin (TBST-BSA) after blocking overnight at 4 °C or 1 h at room temperature (RT) in TBST-BSA.

Techniques: Chromatography, Staining, Control, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Interrogation of glioma immune microenvironment identifies a non-canonical role for microglial Galectin-9 in tumor cell adhesion and phagocytosis

doi: 10.3389/fimmu.2026.1733688

Figure Lengend Snippet: Galectin-9 and Tim-3 expression assessed by cytometry across glioma subtypes. (A, B) Color-coded scatter bar plots represent the relative proportions of Galectin-9 + in A, Tim-3 + in B cells of indicated myeloid and lymphoid cell types across IDH classified primary and recurrent gliomas: NGB ( n = 3), IMP ( n = 14), IMR ( n = 9), IWP ( n = 13), IWR ( n = 12). Statistical differences were determined using Kruskal–Wallis test followed by Dunn’s post-hoc test for multiple comparisons at indicated p values, between NGB versus glioma subtypes, IMP versus IMR, IWP versus IWR, and IMP versus IWP. n.s. = statistically not significant. See also .

Article Snippet: Like any other galectins, cytosolic, secreted and cell surface Galectin-9 protein may lead to pleiotropic functional outcomes, which could be difficult to discern in vivo ( ).

Techniques: Expressing, Cytometry

Journal: Frontiers in Immunology

Article Title: Interrogation of glioma immune microenvironment identifies a non-canonical role for microglial Galectin-9 in tumor cell adhesion and phagocytosis

doi: 10.3389/fimmu.2026.1733688

Figure Lengend Snippet: Galectin-9 expression in tumor and associated leukocytes in glioma. (A) Western blot showing expression of Galectin-9 in flow-sorted CD45 - (tumor), associated leukocytes (CD45 + ), GSC-23, and GSC-28. (B) Representative microscopic image of multiplex IHC stained FFPE tissue sections of primary IDH-mut (top) and IDH-wt (bottom) glioma patients ( n = 5/group). Unmixed images showing expression of Galectin-9 (green), Iba-1 (red), and DAPI (4”,6-diamidino-2-phenylindole) and composite image showing co-expression of Galectin-9 + Iba-1 + MG (crimson, highlighted by arrows) at 40× magnification. Scale bars = 50 μm. (C) Representative microscopic image of multiplex IHC stained FFPE tissue sections of primary IDH-mut (top) and IDH-wt (bottom) glioma patients ( n = 5 per group). Unmixed and composite images showing expression of Galectin-9 (green), Nestin (red), and DAPI (4”,6-diamidino-2-phenylindole) at 40X magnification. Nestin expressing glioma cells do not express Galectin-9 as shown by red and green arrows. Scale bars = 50 μm.

Article Snippet: Like any other galectins, cytosolic, secreted and cell surface Galectin-9 protein may lead to pleiotropic functional outcomes, which could be difficult to discern in vivo ( ).

Techniques: Expressing, Western Blot, Multiplex Assay, Staining

Journal: Frontiers in Immunology

Article Title: Interrogation of glioma immune microenvironment identifies a non-canonical role for microglial Galectin-9 in tumor cell adhesion and phagocytosis

doi: 10.3389/fimmu.2026.1733688

Figure Lengend Snippet: Gene enrichment analysis of Galectin-9 + and Galectin-9 - subpopulations of GAMs. (A) UMAP visualization of MG (left), MAC/MDM = MACs (right) based on differential expression of Galectin-9 gene in IDH-wt glioma patients ( n = 8). Cells are color coded for Galectin-9 expression. (B) Enhanced volcano plot of the variable genes in ( n = 9,372) (top) and Galectin-9 + MACs (bottom) compared to Galectin-9 - counterparts. Gray dots represent genes qualifying average log2FC cutoff of 0.5 and adjusted p -value cutoff of 0.05. The top significant genes for Galectin-9 + GAMs indicated in red. (C) Bubble plot representing the union set of phagocytic markers differentially enriched in Galectin-9 + and Galectin-9 - subpopulations of MG and MACs as indicated. The genes are annotated for their molecular function. The scaled gene expression is shown by the color-bar and the percentage expression by cells is represented by dot size.

Article Snippet: Like any other galectins, cytosolic, secreted and cell surface Galectin-9 protein may lead to pleiotropic functional outcomes, which could be difficult to discern in vivo ( ).

Techniques: Quantitative Proteomics, Expressing, Gene Expression

Journal: Frontiers in Immunology

Article Title: Interrogation of glioma immune microenvironment identifies a non-canonical role for microglial Galectin-9 in tumor cell adhesion and phagocytosis

doi: 10.3389/fimmu.2026.1733688

Figure Lengend Snippet: Glioma cell adhesion was reduced upon Galectin-9 downregulation. (A) Representative microscopic immunofluorescence image showing staining of Galectin-9 (green), Iba-1 (red) and DAPI and their composite expression in merged image of untreated pMG controls (top), Galectin-9 siRNA treated pMG (middle) and siRNA controls (bottom). Scale bars = 90 μm. (B) Diagram showing Ibidi experimental design for pMG/GSC8-11ZsG co-culture assays for adhesion and phagocytosis. (C) Representative microscopic immunofluorescence image showing phase contrast visualization of adhered pMG and GSC8-11ZsG (green) calculated as adhesion ratio when cocultured with untreated pMG controls (top), Galectin-9 siRNA–treated pMG (middle), and siRNA controls (bottom) with GSC8-11ZsG at 2h post-incubation. Scale bars = 40 μm. (D) Scatter dot plots showing corresponding proportions as mean ± SD of % GSC8-11ZsG adhered to indicated pMG from three different fetal donors (pMG-2103, pMG-707, and pMG-1805). Error bars indicate the SD of mean. Statistical differences were determined using Kruskal–Wallis test followed by Dunn’s post-hoc test for multiple comparisons between siRNA treated versus control groups at * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. See also and .

Article Snippet: Like any other galectins, cytosolic, secreted and cell surface Galectin-9 protein may lead to pleiotropic functional outcomes, which could be difficult to discern in vivo ( ).

Techniques: Immunofluorescence, Staining, Expressing, Co-Culture Assay, Incubation, Control

Journal: Frontiers in Immunology

Article Title: Interrogation of glioma immune microenvironment identifies a non-canonical role for microglial Galectin-9 in tumor cell adhesion and phagocytosis

doi: 10.3389/fimmu.2026.1733688

Figure Lengend Snippet: Impaired phagocytic uptake of glioma cell by pMG upon Galectin-9 downregulation. (A) Representative microscopic immunofluorescence image showing phase contrast visualization of pMG, GSC8-11ZsG (green) and pMG/GSC8-11ZsG (merged) exhibiting phagocytosis when cocultured with untreated pMG controls (top), Galectin-9 siRNA–treated pMG (middle) and siRNA controls (bottom) with GSC8-11ZsG at 2h post-incubation. Scale bars = 60 μm. White boxes shows magnified image that depicts amount of GSC8-11ZsG by pMG. (B) Scatter dot plots showing mean proportions ± SD of pMG (pMG-2103, pMG-707, and pMG-1805) that phagocytosed GSC8-11ZsG (phagocytosis ratio) in untreated control, treated Galectin-9 siRNA, and siRNA control experimental conditions when co-cultured with GSC8-11ZsG. Error bars indicate the SD of mean. Statistical differences were determined using Kruskal–Wallis test followed by Dunn’s post-hoc test for multiple comparisons between siRNA-treated versus control groups at ** p < 0.01, **** p < 0.0001. (C) , Scatter dot plots showing mean proportions ± SD of pMG (pMG-2103, pMG-707, and pMG-1805) that phagocytosed GSC8-11ZsG (phagocytosis ratio) in untreated control, treated Galectin-9 neutralization Ab (MAb-13), and IgG control experimental conditions when co-cultured with GSC8-11ZsG. Error bars indicate the SD of mean. Statistical differences were determined using Kruskal–Wallis test followed by Dunn’s post-hoc test for multiple comparisons between siRNA-treated versus control groups at *** p < 0.001, **** p < 0.0001. See also .

Article Snippet: Like any other galectins, cytosolic, secreted and cell surface Galectin-9 protein may lead to pleiotropic functional outcomes, which could be difficult to discern in vivo ( ).

Techniques: Immunofluorescence, Incubation, Control, Cell Culture, Neutralization