Journal: bioRxiv

Article Title: Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth

doi: 10.1101/2023.10.05.561003

Figure Lengend Snippet: Galectin-9 inhibits mycobacterial growth directly . A. Profile of Mtb H37Rv (Rv) grown at 37°C in Middlebrook 7H9 liquid medium with different concentration of Galectin-9 (Gal9, 0, 0.01, 0.1, 1, 10 μg/mL). Growth curve was measured using a Bioscreen Growth Curve Instrument. Optical density was measured at absorbance at 600 nm every 2 h. B. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with10μg/mL galectin-9 (Gal9) or inactivated galectin-9 (Gal9 HK, heat killed at 95 [for 5 min). C. CFU of Mtb H37Rv (Rv) on Middlebrook 7H10 solid medium with or without 10μg/mL Galectin-9 (Gal9). Cultures were grown at 37°C for 4-8 weeks. D. Growth profile of Mycobacterium smegmatis (MS) in Middlebrook 7H9 liquid medium with different concentrations of Galectin-9 (Gal9, 0, 0.01, 0.1, 1 μg/mL). E. Concentrations of galectin-9 in sera of healthy donors (n = 40) and active TB patients (n = 40). F. Confocal microscopy of M. bovis BCG-DsRed (BCG-DsRed, red) and Galectin[9 (Anti-Gal9, green) in THP-1 cells. Nuclei was stained with DAPI (blue). G. Percent of cells with galectin-9 positive (gal9 + ) BCG in total infected THP-1 cells. Symbols indicate colocalization ratio of at least 12 fields in each experiment. H. Confocal microscopy of Mtb H37Rv-GFP (Rv-GFP, green) and Galectin[9 (Anti-Gal9, red) in THP-1 cells. Nuclei were stained with DAPI (blue). I. Percent of cells with galectin9 positive (gal9 + ) Mtb H37Rv in total infected THP-1 cells. Symbols indicate colocalization ratio of at least 12 fields in each experiment. Data are shown as mean ± SD, n = 3 biologically independent experiments performed in triplicate (A-D). Data are representative of three independent experiments with similar results (F and H). Two-tailed unpaired Student’s t test (A-D, G, and I) or Mann-Whitney U test (E). P < 0.05 was considered statistically significant.

Article Snippet: Cells were stained with the anti-galectin-9 antibody (Cell Signaling Technology, Cat#54330, RRID:AB_2799456), antibodies at a dilution of 1:200 in 5% BSA in PBS overnight at 4[°C and then incubated with Alexa Fluor 488 or 555 conjugated secondary antibodies (Thermo Fisher Scientific, Cat# A-11008; RRID: AB_143165; Cat# A32732, RRID:AB_2633281) at a dilution of 1:1000 for 2[h at R.T..

Techniques: Concentration Assay, Confocal Microscopy, Staining, Infection, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth

doi: 10.1101/2023.10.05.561003

Figure Lengend Snippet: A. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and lactose (1 μg/mL). B. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and D-glucose (10 μg/mL). C. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and AG (1 μg/mL). D. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with 1μg/mL CRD1 or CRD2 of galectin-9. Data are shown as mean ± SD, n = 3 biologically independent experiments performed in triplicate (A-D). Two-tailed unpaired Student’s t test (A-D). P < 0.05 was considered statistically significant.

Article Snippet: Cells were stained with the anti-galectin-9 antibody (Cell Signaling Technology, Cat#54330, RRID:AB_2799456), antibodies at a dilution of 1:200 in 5% BSA in PBS overnight at 4[°C and then incubated with Alexa Fluor 488 or 555 conjugated secondary antibodies (Thermo Fisher Scientific, Cat# A-11008; RRID: AB_143165; Cat# A32732, RRID:AB_2633281) at a dilution of 1:1000 for 2[h at R.T..

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth

doi: 10.1101/2023.10.05.561003

Figure Lengend Snippet: A. Morphologic characteristics for Mtb H37Rv strain grown in liquid culture with or without anti-AG mAbs (1 μg/mL) observed by ×[2 magnifier. B. Bacterial shape of Mtb H37Rv strain treated as in (A) observed by acid fast staining under a Leica DM2500 microscope using the 100× oil microscopy. EMB, Ethambutol. Scale bar, 20μm. C. Ultrastructural morphology of Mtb H37Rv treated as in (A) analyzed by transmission electron microscopy (TEM). The cell wall was labeled with red arrows. D. Cell wall thickness of bacteria in (C). E. Schematic presentation of Mtb growth arrest by Galectin-9 or anti-AG antibodies. Data are representative of three independent experiments with similar results (A, B and C). Data are means ± SD of 11 bacteria, representatives of three independent experiments (D). Two-tailed unpaired Student’s t test (D). P < 0.05 was considered statistically significant.

Article Snippet: Cells were stained with the anti-galectin-9 antibody (Cell Signaling Technology, Cat#54330, RRID:AB_2799456), antibodies at a dilution of 1:200 in 5% BSA in PBS overnight at 4[°C and then incubated with Alexa Fluor 488 or 555 conjugated secondary antibodies (Thermo Fisher Scientific, Cat# A-11008; RRID: AB_143165; Cat# A32732, RRID:AB_2633281) at a dilution of 1:1000 for 2[h at R.T..

Techniques: Staining, Microscopy, Transmission Assay, Electron Microscopy, Labeling, Bacteria, Two Tailed Test

Journal: The Journal of pathology

Article Title: CD206-positive myeloid cells bind galectin-9 and promote a tumor-supportive microenvironment

doi: 10.1002/path.5093

Figure Lengend Snippet: (A) Pre-incubation with galectin-9 (light grey) decreases the ability of anti-CD206 to bind CD68+CD206+ macrophages. (B) Pre-incubation with anti-CD206 antibody (light grey) decreases the ability of galectin-9 protein to bind CD68+CD206+ macrophages. (C) Co-localization of CD206 and galectin-9 on human M2 macrophages. (D) Western blot of CD206 after galectin-9 immunoprecipitation using M2 polarized THP-1 cells. (E) Western blot of galectin-9 after CD206 immunoprecipitation using M2 polarized THP-1 cells. (F) Protein-protein binding by Blitz indicates a dissociation constant of 2.8 × 10−7 M. Data are representative of four different experiments.

Article Snippet: Immunohistochemistry was performed with mouse monoclonal antibodies against galectin-9 (clone 1G3, ab153673, Abcam, Cambridge, United Kingdom) and CD206 (IgG2B clone # 685645, R&D systems, Minneapolis, MN, United States).

Techniques: Incubation, Western Blot, Immunoprecipitation, Protein Binding

Journal: The Journal of pathology

Article Title: CD206-positive myeloid cells bind galectin-9 and promote a tumor-supportive microenvironment

doi: 10.1002/path.5093

Figure Lengend Snippet: (A) Secretion of FGF-2 after incubation with galectin-9. (B) Concentration of HGF in the supernatant of cells treated with galectin-9. (C) VEGF concentration in culture media after galectin-9. (D) Secretion of MDC after galectin-9 incubation. (E) Culture media concentrations of MCP-1 after galectin-9 treatment. (F) Media concentration of IP-10 after galectin-9 treatment. Data presented as average ± SD; * indicates p < 0.05. Black: untreated, grey: 2 μg/ml galectin-9; n = 6.

Article Snippet: Immunohistochemistry was performed with mouse monoclonal antibodies against galectin-9 (clone 1G3, ab153673, Abcam, Cambridge, United Kingdom) and CD206 (IgG2B clone # 685645, R&D systems, Minneapolis, MN, United States).

Techniques: Incubation, Concentration Assay

Journal: The Journal of pathology

Article Title: CD206-positive myeloid cells bind galectin-9 and promote a tumor-supportive microenvironment

doi: 10.1002/path.5093

Figure Lengend Snippet: (A) Changes in classical and non-classical monocyte subtypes after galectin-9 treatment. (B) CCR2 receptor is slightly downregulated on classical monocytes after galectin-9. (C) Galectin-9 significantly increased CD14+CD16+CX3CR1 populations. (D) CD206 is expressed at a higher level after monocytes are treated with galectin-9. Data presented as mean fluorescence intensities (MFI) or percentage of positive cells ± s.d. * indicates p < 0.05. White: untreated, grey: 2 μg/ml galectin-9; n=10.

Article Snippet: Immunohistochemistry was performed with mouse monoclonal antibodies against galectin-9 (clone 1G3, ab153673, Abcam, Cambridge, United Kingdom) and CD206 (IgG2B clone # 685645, R&D systems, Minneapolis, MN, United States).

Techniques: Fluorescence

Journal: The Journal of pathology

Article Title: CD206-positive myeloid cells bind galectin-9 and promote a tumor-supportive microenvironment

doi: 10.1002/path.5093

Figure Lengend Snippet: (A) Examples of galectin-9 tumor cell expression as assessed by H-score. (B) Representative groups with different levels of intratumoral CD206+ macrophage staining: 0 = negative (n = 3), 1 = minimal (n = 81), 2 = moderate (n = 76), 3 = dense (n = 20). (C) Tumors highly positive for galectin-9 are associated with highest staining of CD206. (D) Kaplan-Meier curve of high (groups 2–3) versus low (groups 0–1) CD206 expression with confidence intervals (p = 0.019). (E) Kaplan-Meier curve of high (≥65) versus low (<65) galectin-9 tumor expression with confidence intervals (p = 0.3).

Article Snippet: Immunohistochemistry was performed with mouse monoclonal antibodies against galectin-9 (clone 1G3, ab153673, Abcam, Cambridge, United Kingdom) and CD206 (IgG2B clone # 685645, R&D systems, Minneapolis, MN, United States).

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Upregulation of the Tim-3/Galectin-9 Pathway of T Cell Exhaustion in Chronic Hepatitis B Virus Infection

doi: 10.1371/journal.pone.0047648

Figure Lengend Snippet: PBMC derived from HLA A2+ patients with CHB were stained with a panel of HLA-A2/HBV multimers and then were stimulated overnight with a pool of HBV peptides of matched specificity to the multimers, followed by intracellular staining for IFN-γor TNF-α. (a) Representative histograms showing levels of Tim-3 (black line) or isotype binding (grey shading) on CD8 T cells binding HLA-A2/HBV peptide multimers or producing IFN-γ upon encounter with HBV peptides. (b) Compiled data from 10 patients with CHB. (c) Tim-3 expression on CD8 T cells binding HLA-A2/HBV peptide multimers or producing TNFa upon stimulation with HBV peptides. (d) FACS plots and (e) summary data showing the induction of caspases (FLICA) and 7AAD in CD8 and CD4 T cells with or without the addition of galectin-9. Active caspases, indicating apoptosis, were determined using a fluorescent-labelled inhibitor of polycaspases (FAM-VAD-FMK, FLICA), and death was identified by 7AAD stain. Early apoptotic events are indicated in the lower right quadrant (FLICA+7AAD−), late apoptotic events in the right upper quadrant (FLICA+7AAD+) and necrotic cells in the left upper quadrant (7AAD+FLICA-). ‘Total death’ was estimated by summing events in these 3 quadrants.

Article Snippet: Immunostaining of cryopreserved sections from HBV infected liver biopsies was performed using a combination of the following primary antibodies Galectin-9 goat polyclonal IgG (R & D Systems) and CD68 mouse monoclonal (KP1, Abcam).

Techniques: Derivative Assay, Staining, Binding Assay, Expressing

Journal: PLoS ONE

Article Title: Upregulation of the Tim-3/Galectin-9 Pathway of T Cell Exhaustion in Chronic Hepatitis B Virus Infection

doi: 10.1371/journal.pone.0047648

Figure Lengend Snippet: Immunohistochemistry of a section from a cryopreserved CHB liver biopsy stained with a polyclonal galectin-9 antibody (shown in brown) at 10X and 40X magnification. (b ) Immunofluorescence of a section from a cryopreserved CHB liver biopsy stained with DAPI (blue, left panel), anti-CD68 mAb (green, central panel) and galectin 9 polyclonal antibody (red, right panel). Double positive staining is indicated in yellow in the lower panel. (c) Kupffer cells were isolated from 4 liver explants and stained with CD14, CD68 and galectin-9 or its isotype. Representative histograms show galectin-9 staining on CD14+CD68+ fraction compared to isotype-matched control. (d) Galectin-9 levels were analysed in the serum of 10 healthy subjects and 42 CHB patients: 16 with ALT<50, 17 with ALT 50–100 and 9 with ALT >100 (mean and SEM shown).

Article Snippet: Immunostaining of cryopreserved sections from HBV infected liver biopsies was performed using a combination of the following primary antibodies Galectin-9 goat polyclonal IgG (R & D Systems) and CD68 mouse monoclonal (KP1, Abcam).

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Isolation

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: Galectin 9 , REA435 , 50 , 130-124-237 , PE (APC) , Miltenyi Biotec.

Techniques: Imaging