Journal: American journal of physiology. Cell physiology

Article Title: ADAM17-induced shedding of glypican-1 as a mechanism of impaired endothelial shear stress mechanotransduction

doi: 10.1152/ajpcell.00794.2025

Figure Lengend Snippet: A: Brachial artery flow-mediated dilation (FMD, expressed as percent change in arterial diameter), plasma neuraminidase and ADAM17 activities (expressed as fold difference from healthy participants (males, n=12; females, n=18; open squares and triangles, respectively) and participants with T2D (males, n=26; females, n= 21; solid squares and triangles, respectively). B: FMD and sodium nitroprusside (SNP)-induced vasodilation in mouse-isolated femoral arteries (n=5-6/group). Arteries were preconstricted with phenylephrine. C: Representative images (left panel) and quantification (right panel) of endothelial glypican-1 content on the luminal surface of the endothelium in isolated femoral arteries from wild-type (WT) and db/db mice (n=8/group). Data are presented as means ± SE. Mann-Whitney test (A). Two-way repeated-measures ANOVA (B). Two-tailed unpaired t test (C). * P <0.05 vs. healthy participants (A) or WT mice (B-C).

Article Snippet: Plasma neuraminidase activity was measured using a buffer containing sodium acetate, pH 4.5, TritonX-100, and 12′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (EM05195, BioSynth), as previously described ( 12 ).

Techniques: Shear, Clinical Proteomics, Isolation, MANN-WHITNEY, Two Tailed Test

Journal: American journal of physiology. Cell physiology

Article Title: ADAM17-induced shedding of glypican-1 as a mechanism of impaired endothelial shear stress mechanotransduction

doi: 10.1152/ajpcell.00794.2025

Figure Lengend Snippet: Endothelial cells and isolated arteries were exposed to neuraminidase (Neu, 100 mU/mL) vs. vehicle for 1 hr. Incubated arteries were treated intraluminally. A: Extracellular sialic acid was quantified using WGA (n=11-12/condition, including representative images, scale bar=80 μm); MAA/MAL I+II (n=11-12/condition, including representative images, scale bar=40 μm). B-C: Fluo4 was used to detect changes in intracellular Ca 2+ (F1/F0) in cells (plate reader fluorescence n=8-13/condition; fluorescence microscopy (n=5-6/condition); including representative images from the 60 min timepoint, scale bar=50 μm) and isolated mesenteric arteries from wild-type male mice (C57BL/6J, n=6-7/condition; including representative images from the 60 min timepoint, scale bar=50 μm). D: Phosphatidylserine (PS) externalization in the presence or absence of Ca 2+ ionophore (A23187, 10 μM, 1 hr) and ANO6 inhibitor (ANO6-I, A6-001,10 μM; n=12/condition). Data are presented as means ± SE. Two-tailed unpaired t test (A-B). Two-way repeated-measures ANOVA (C). One-way ANOVA (D). *, # P <0.05 vs. respective controls.

Article Snippet: Plasma neuraminidase activity was measured using a buffer containing sodium acetate, pH 4.5, TritonX-100, and 12′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (EM05195, BioSynth), as previously described ( 12 ).

Techniques: Isolation, Incubation, Fluorescence, Microscopy, Two Tailed Test

Journal: American journal of physiology. Cell physiology

Article Title: ADAM17-induced shedding of glypican-1 as a mechanism of impaired endothelial shear stress mechanotransduction

doi: 10.1152/ajpcell.00794.2025

Figure Lengend Snippet: Endothelial cells were exposed to neuraminidase (Neu, 100 mU/mL) vs. vehicle for 1 hr. A: PS externalization (n=20-24/condition); B: ADAM17 activity (n=12/condition); C: shedding of glypican-1 (cell culture supernatant relative to lysate, n=12/condition, including representative blots. Data are presented as means ± SE. Two-tailed unpaired t test (A-C). *P <0.05 vs. respective controls.

Article Snippet: Plasma neuraminidase activity was measured using a buffer containing sodium acetate, pH 4.5, TritonX-100, and 12′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (EM05195, BioSynth), as previously described ( 12 ).

Techniques: Activity Assay, Cell Culture, Two Tailed Test

Journal: American journal of physiology. Cell physiology

Article Title: ADAM17-induced shedding of glypican-1 as a mechanism of impaired endothelial shear stress mechanotransduction

doi: 10.1152/ajpcell.00794.2025

Figure Lengend Snippet: A: Schematic representation of the experimental protocol for intraluminally treated isolated mesenteric arteries from wild-type mice. From each mouse, four mesenteric arteries were isolated and studied as shown. B: Effects of 1 hr incubation with neuraminidase (Neu, 100 mU/mL) vs. vehicle (1:250 DMSO) on FMD and SNP-induced vasodilation (n=9/condition). C-D: Mesenteric arteries from the same mice as in B were pre-incubated with the ANO6 or ADAM17 inhibitors, or vehicle (1:250 DMSO). After a 1 hr pre-incubation with the inhibitor treatments, all arteries were exposed to either Neu (100 mU/mL) or the same vehicle for an additional 1 hr. (C) Effects of 1 hr incubation with Neu in the presence or absence of an ANO6 inhibitor (ANO6-i; A6-001, 10 μM) on FMD and SNP-induced vasodilation (n=9/condition). (D) Effects of 1 hr incubation with Neu in the presence or absence of an ADAM17 inhibitor (TAPI-0, 50 μM) on FMD and SNP-induced vasodilation (n=9/condition). Vessels were preconstricted with phenylephrine. Panels B-D share the same Neu dataset. Data are presented as means ± SE. Two-way repeated-measures ANOVA. * P <0.05 vs. respective controls.

Article Snippet: Plasma neuraminidase activity was measured using a buffer containing sodium acetate, pH 4.5, TritonX-100, and 12′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (EM05195, BioSynth), as previously described ( 12 ).

Techniques: Isolation, Inhibition, Incubation

Journal: American journal of physiology. Cell physiology

Article Title: ADAM17-induced shedding of glypican-1 as a mechanism of impaired endothelial shear stress mechanotransduction

doi: 10.1152/ajpcell.00794.2025

Figure Lengend Snippet: Endothelial cells were exposed to Neu (100 mU/mL) for 1 hr with or without an ANO6-i (A6-001,10 μM). ANO6-i was added 1 hr before Neu. A: PS externalization (n=9/condition); B: ADAM17 activity (n=12/condition); C: shedding of glypican-1 (cell culture supernatant relative to lysate, n=7-8/condition, including representative blots) in endothelial cells. Data are presented as means ± SE. Two-tailed unpaired t test (A-C). *P <0.05 vs. respective controls.

Article Snippet: Plasma neuraminidase activity was measured using a buffer containing sodium acetate, pH 4.5, TritonX-100, and 12′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (EM05195, BioSynth), as previously described ( 12 ).

Techniques: Inhibition, Activity Assay, Cell Culture, Two Tailed Test

Journal: American journal of physiology. Cell physiology

Article Title: ADAM17-induced shedding of glypican-1 as a mechanism of impaired endothelial shear stress mechanotransduction

doi: 10.1152/ajpcell.00794.2025

Figure Lengend Snippet: Endothelial cells were exposed to Neu (100 mU/mL) for 1 hr with or without an ADAM17 inhibitor (TAPI-0, 50 μM). TAPI-0 was added 1 hr before Neu. A: PS externalization (n=11-12/condition); B: ADAM17 activity (n=11-23/condition); C: shedding of glypican-1 (cell culture supernatant relative to lysate, n=11-12/condition, including representative blots) in endothelial cells. Data are presented as means ± SE. Two-tailed unpaired t test (A-C). *P <0.05 vs. respective controls.

Article Snippet: Plasma neuraminidase activity was measured using a buffer containing sodium acetate, pH 4.5, TritonX-100, and 12′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (EM05195, BioSynth), as previously described ( 12 ).

Techniques: Inhibition, Activity Assay, Cell Culture, Two Tailed Test

Journal: American journal of physiology. Cell physiology

Article Title: ADAM17-induced shedding of glypican-1 as a mechanism of impaired endothelial shear stress mechanotransduction

doi: 10.1152/ajpcell.00794.2025

Figure Lengend Snippet: Neuraminidase, which is elevated in the circulation of individuals with type 2 diabetes, induces an increase in endothelial intracellular Ca 2+ (1). This Ca 2+ increase is likely the stimulus for activation of Anoctamin-6 (ANO6), which flips phosphatidylserine (PS) to the outer leaflet of the plasma membrane (2). The PS head group is negatively charged. Loss of plasma membrane asymmetry by flipped PS causes its interaction with the membrane proximal domain of ADAM17, which presents a cationic PS-binding motif. This interaction causes ADAM17 to bend to the appropriate position for substrate shedding, including mechanosensitive structures such as glypican-1 (3). Loss of glypican-1 likely contributes to impaired shear stress mechanotransduction (4).

Article Snippet: Plasma neuraminidase activity was measured using a buffer containing sodium acetate, pH 4.5, TritonX-100, and 12′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (EM05195, BioSynth), as previously described ( 12 ).

Techniques: Activation Assay, Clinical Proteomics, Membrane, Binding Assay, Shear

Journal: Cancers

Article Title: Pro-Inflammatory Cytokines Transactivate Glycosylated Cytokine Receptors on Cancer Cells to Induce Epithelial–Mesenchymal Transition to the Metastatic Phenotype

doi: 10.3390/cancers17071234

Figure Lengend Snippet: Figure 1. Cytokine receptor binding is implicated in the Neu-1-MMP-9-GPCR crosstalk. The binding of a cytokine to its receptor, a receptor tyrosine kinase (RTK), induces conformational changes in the associated GPCR, which in turn activates MMP-9 through the Gα subunit. This change activates Neu-1 by releasing the elastin-binding protein (EBP) from its complex with Neu-1 and protective protein cathepsin A (PPCA). Neu-1 cleaves α-2,3 sialic acid from the terminal glycosyl residue of the receptor, removing steric hindrance, which in turn induces the cytokine-dependent signal from the cytokine-binding receptor. Inhibitors of various components of this signaling paradigm are oseltamivir phosphate (OP; inhibitor of Neu-1), MMP9i (MMP-9 inhibitor), and BIM 23127 (NMBR inhibitor). Abbreviations: RTK: receptor tyrosine kinase; GPCR: G-protein-coupled receptor; MMP-9: matrix metalloprotease-9; Neu-1: neuraminidase-1; PPCA: protective protein cathepsin A; EBP: elastin-binding protein; HGF: hepatocyte growth factor; c-MET: HGF receptor. Citation. Modified in part from Research and Reports in Biochemistry 2013:3 17–30© 2013 Abdulkhalek et al., https://doi.org/10.2147/RRBC.S28430 (accessed on 7 January 2013) [29], publisher and licensee Dove Medical Press Ltd. This is an open-access article that permits unrestricted non-commercial use, provided the original work is properly cited.

Article Snippet: To measure neuraminidase activity, 2-(4-methylumbelliferyl)-α-D-Nacetylneuraminic acid (98% pure 4-MUNANA; Biosynth International Inc., Itasca, IL, USA), a sialidase substrate with excitation at 365–380 nm and emission at 445–454 nm, was used.

Techniques: Binding Assay, Residue, Modification

Journal: Cancers

Article Title: Pro-Inflammatory Cytokines Transactivate Glycosylated Cytokine Receptors on Cancer Cells to Induce Epithelial–Mesenchymal Transition to the Metastatic Phenotype

doi: 10.3390/cancers17071234

Figure Lengend Snippet: Figure 2. Cytokines increase sialidase activity in PANC-1 cells, while inhibitors of signaling paradigm OP, MMP9i, and BIM23 reverse cytokine activity. Pancreatic cancer cells (PANC-1) were treated with either TGFβ-1 (4.0 × 10−3 µg/mL) (A–D), IL-6 (4.1 × 10−5 µg/mL) (E–H), HGF (5.97 × 10−4 µg/mL) (I–L), and cells treated with inhibitors alone (M) for one minute. 4-MUNANA substrate was added to live cells to measure sialidase activity (emission 450 nm, excitation 365 nm). The addition of (A–D) TGFβ-1, (E–H) IL-6, and (I–L) HGF increased the sialidase fluorescence compared to the un- treated control. In contrast, the use of inhibitors of Neu-1 (OP), MMP-9 (MMP9i), and NMBR/GPCR (BIM23) significantly decreased sialidase fluorescence in TGFβ-1 (D), IL-6 (H), and HGF (L) treatment groups. The mean ± SEM (biological replicates: 5, technical replicates: 50) fluorescence density of sialidase activity demonstrates a highly significant (p < 0.0001) downregulation of sialidase activity with all three inhibitors. Abbreviations: TGFβ-1: transforming growth factor beta-1; IL-6: interleukin- 6; HGF: hepatocyte growth factor; OP: oseltamivir phosphate; MMP9i: MMP9 inhibitor; BIM23: BIM-23127. **** p < 0.0001.

Article Snippet: To measure neuraminidase activity, 2-(4-methylumbelliferyl)-α-D-Nacetylneuraminic acid (98% pure 4-MUNANA; Biosynth International Inc., Itasca, IL, USA), a sialidase substrate with excitation at 365–380 nm and emission at 445–454 nm, was used.

Techniques: Activity Assay, Fluorescence, Control

Journal: Cancers

Article Title: Pro-Inflammatory Cytokines Transactivate Glycosylated Cytokine Receptors on Cancer Cells to Induce Epithelial–Mesenchymal Transition to the Metastatic Phenotype

doi: 10.3390/cancers17071234

Figure Lengend Snippet: Figure 3. Cytokines increase sialidase activity in colorectal SW620 cells, while inhibitors of signaling paradigm OP, MMP9i, and BIM23 reverse cytokine activity. Colorectal cancer cells (SW620) were treated with either TGFβ-1 (4.0 × 10−3 µg/mL), IL-6 (4.1 × 10−5 µg/mL), or HGF (5.97 × 10−4 µg/mL). 4- MUNANA substrate was added to live cells to measure sialidase activity (emission 450 nm, excitation 365 nm). The addition of (A) TGFβ-1, (B) IL-6, and (C) HGF increased the sialidase fluorescence compared to the untreated control. In contrast, the use of inhibitors of Neu-1 (OP), MMP-9 (MMP9i), and NMBR/GPCR (BIM23) significantly decreased sialidase fluores- cence in TGFβ-1, IL-6, and HGF treatment groups (D–F). The mean ± SEM (biological replicates: 5, technical replicates: 50) fluorescence density of sialidase activity (G–I) demonstrates a highly significant (p < 0.0001) downregulation of sialidase activity with all three inhibitors. Abbreviations: TGFβ-1: transforming growth factor beta-1; IL-6: interleukin-6; HGF: hepatocyte growth factor; OP: oseltamivir phosphate; MMP9i: MMP9 inhibitor; BIM23: BIM-23127. **** p < 0.0001.

Article Snippet: To measure neuraminidase activity, 2-(4-methylumbelliferyl)-α-D-Nacetylneuraminic acid (98% pure 4-MUNANA; Biosynth International Inc., Itasca, IL, USA), a sialidase substrate with excitation at 365–380 nm and emission at 445–454 nm, was used.

Techniques: Activity Assay, Fluorescence, Control

Journal: Cancers

Article Title: Pro-Inflammatory Cytokines Transactivate Glycosylated Cytokine Receptors on Cancer Cells to Induce Epithelial–Mesenchymal Transition to the Metastatic Phenotype

doi: 10.3390/cancers17071234

Figure Lengend Snippet: Figure 4. Cytokines increase sialidase activity in breast MCF-7 cells, while inhibitors of signaling paradigm OP, MMP9i, and BIM23 reverse cytokine activity. Breast cancer cells (MCF-7) were treated with either TGFβ-1 (4.0 × 10−3 µg/mL), IL-6 (4.1 × 10−5 µg/mL), or HGF (5.97 × 10−4 µg/mL). 4-MUNANA substrate was added to live cells to measure sialidase activity (emission 450 nm, excitation 365 nm). The addition of (A) TGFβ-1, (B) IL-6, and (C) HGF increased the sialidase fluorescence compared to the untreated control. In contrast, the use of inhibitors of Neu-1 (OP), MMP- 9 (MMP9i), and NMBR/GCPR (BIM23) significantly decreased sialidase fluorescence in TGFβ-1, IL-6, and HGF treatment groups (D–F). The mean ± SEM (biological replicates: 5, technical replicates: 50) fluorescence density of sialidase activity (G–I) demonstrates a highly significant (p < 0.0001) downregulation of sialidase activity with all three inhibitors. Abbreviations: TGFβ-1: transforming growth factor beta-1; IL-6: interleukin-6; HGF: hepatocyte growth factor; OP: oseltamivir phosphate; MMP9i: MMP9 inhibitor; BIM23: BIM-23127. **** p < 0.0001.

Article Snippet: To measure neuraminidase activity, 2-(4-methylumbelliferyl)-α-D-Nacetylneuraminic acid (98% pure 4-MUNANA; Biosynth International Inc., Itasca, IL, USA), a sialidase substrate with excitation at 365–380 nm and emission at 445–454 nm, was used.

Techniques: Activity Assay, Fluorescence, Control

Journal: Cancers

Article Title: Pro-Inflammatory Cytokines Transactivate Glycosylated Cytokine Receptors on Cancer Cells to Induce Epithelial–Mesenchymal Transition to the Metastatic Phenotype

doi: 10.3390/cancers17071234

Figure Lengend Snippet: Figure 5. Receptors of TGFβ-1, IL-6, and HGF colocalize with neuraminidase-1 in PANC-1 cancer cells. Cells were fixed, permeabilized, blocked, and immunostained with the specific antibodies. PANC-1 cells were treated with Alexa Fluor™594-conjugated antibodies for the cytokine receptors TGFβR, IL- 6R, and HGFR/c-MET and Alexa Fluor™488-conjugated Neu-1 antibody. (A) Merging of fluorescent images acquired from Zeiss M2 epi-fluorescent microscope (40× objective magnification) shows areas of colocalization through yellow fluorescence. Pearson correlation coefficient (R-value) measured a significant positive correlation (0.69 < r < 0.74) between the cytokine receptors (B) TGFβR, (C) IL-6R, and (D) HGFR/c-MET and Neu-1 (biological replicates: 4, technical replicates: 1). Abbreviations: TGFβR: transforming growth factor beta receptor; IL-6R: interleukin-6 receptor; HGFR/c-MET: hepatocyte growth factor receptor; Neu-1: neuraminidase-1.

Article Snippet: To measure neuraminidase activity, 2-(4-methylumbelliferyl)-α-D-Nacetylneuraminic acid (98% pure 4-MUNANA; Biosynth International Inc., Itasca, IL, USA), a sialidase substrate with excitation at 365–380 nm and emission at 445–454 nm, was used.

Techniques: Microscopy, Fluorescence

Journal: Cancers

Article Title: Pro-Inflammatory Cytokines Transactivate Glycosylated Cytokine Receptors on Cancer Cells to Induce Epithelial–Mesenchymal Transition to the Metastatic Phenotype

doi: 10.3390/cancers17071234

Figure Lengend Snippet: Figure 6. Receptors of TGFβ-1, IL-6, and HGF moderately colocalize with neuraminidase-1 in SW620 cancer cells. Cells were fixed, permeabilized, blocked, and immunostained with the specific antibodies. SW620 cells were treated with Alexa Fluor™594-conjugated antibodies for the cytokine receptors TGFβR, IL-6R, and HGFR/c-MET and Alexa Fluor™488-conjugated Neu-1 antibody. (A) Merging of fluorescent images acquired from Zeiss M2 epi-fluorescent microscope (40× objective magnification) shows areas of colocalization through yellow fluorescence. Pearson correlation coefficient (R-value) measured a moderate correlation (0.55 < r < 0.60) between the cytokine receptors (B) TGFβR, (C) IL-6R, and (D) HGFR/c-MET and Neu-1 (biological replicates: 4, technical replicates: 1). Abbreviations: TGFβR: transforming growth factor beta receptor; IL-6R: interleukin-6 receptor; HGFR/c-MET: hepatocyte growth factor receptor; Neu-1: neuraminidase-1.

Article Snippet: To measure neuraminidase activity, 2-(4-methylumbelliferyl)-α-D-Nacetylneuraminic acid (98% pure 4-MUNANA; Biosynth International Inc., Itasca, IL, USA), a sialidase substrate with excitation at 365–380 nm and emission at 445–454 nm, was used.

Techniques: Microscopy, Fluorescence

Journal: Cancers

Article Title: Pro-Inflammatory Cytokines Transactivate Glycosylated Cytokine Receptors on Cancer Cells to Induce Epithelial–Mesenchymal Transition to the Metastatic Phenotype

doi: 10.3390/cancers17071234

Figure Lengend Snippet: Figure 7. Receptors of TGFβ-1, IL-6, and HGF markedly colocalize with neuraminidase-1 in MCF- 7 cancer cells. Cells were fixed, permeabilized, blocked, and immunostained with the specific antibodies. MCF-7 cells were treated with Alexa Fluor™594-conjugated antibodies for the cytokine receptors TGFβR, IL-6R, and HGFR/c-MET and Alexa Fluor™488-conjugated Neu-1 antibody. (A) Merging of fluorescent images acquired from Zeiss M2 epi-fluorescent microscope (40× objective magnification) shows areas of colocalization (yellow) fluorescence. Pearson correlation coefficient (R-value) measured a strong positive correlation (0.81 < r < 0.92) between the cytokine receptors (B) TGFβR, (C) IL-6R, and (D) HGFR/c-MET and Neu-1 (biological replicates: 4, technical replicates: 1). Abbreviations: TGFβR: transforming growth factor beta receptor; IL-6R: interleukin-6 receptor; HGFR/c-MET: hepatocyte growth factor receptor; Neu-1: neuraminidase-1.

Article Snippet: To measure neuraminidase activity, 2-(4-methylumbelliferyl)-α-D-Nacetylneuraminic acid (98% pure 4-MUNANA; Biosynth International Inc., Itasca, IL, USA), a sialidase substrate with excitation at 365–380 nm and emission at 445–454 nm, was used.

Techniques: Microscopy, Fluorescence

Journal: Cellular and Molecular Immunology

Article Title: Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2

doi: 10.1038/s41423-024-01142-0

Figure Lengend Snippet: Reduced suppressive function of MDSCs lacking Siglec-E upon sialidase or Siglec-E blocking antibody treatment. A Experimental setup: Depletion of Ly6G-positive cells in SigE ΔLysM mice and SigE WT littermates bearing B16F10 tumors using a depleting antibody. Mice were injected up to 6 times (gray arrow) with the anti-Ly6G depletion antibody starting 1 day before subcutaneous B16F10 tumor injection (black arrow). Tumor growth and survival were monitored. B Kaplan‒Meier survival curves or ( C ) tumor growth curves from pooled experiments from ( A ). n = 9–11 mice per group . D Experimental setup for assessing the effect of the DR5 antibody on SigE ΔLysM mice and SigE WT littermates subcutaneously injected with B16F10 tumors. E Kaplan‒Meier survival curves and ( F ) tumor growth curves from ( D ) with n = 6–7 mice per group . G Experimental setup for assessing the suppressive effect of Gr1 + CD11b + (MDSC) cells on naïve CD3 + (T cell) cells. MDSCs were isolated from the spleens of B16F10 tumor-bearing SigE ΔLysM mice and SigE WT littermates. T cells were isolated from naïve littermates and stained with CellTrace Violet (CTV) to track T-cell proliferation by flow cytometry. Stained T cells were cocultured with MDSCs for 48 h in the presence of aCD3, aCD28 and IL-2. MDSCs were used immediately or pretreated with sialidase. A Siglec-E blocking antibody or rat IgG2a, κ isotype control was added to the cocultures as indicated. H Percentage of proliferating CD8 + T cells cocultured without MDSCs, ( I ) with MDSCs from SigE WT mice or ( J ) MDSCs from SigE ΔLysM mice. Exemplary results for each untreated condition are shown on the right, indicating untreated T cells alone (J, gray), SigE WT (K, green), and SigE ΔLysM (L, pink). n = 3–9 mice per condition . The data are presented as the mean ± SD or SEM ( C , F ). Multiple paired t tests were used. For survival analysis, the log-rank test was used, followed by the Šidák correction for multiple comparisons. Tumor growth was compared by mixed-effects analysis followed by Bonferroni’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: Additionally, viral sialidase (active H1N1 ; Sino Biological) and bacterial sialidase ( Arthrobacter ureafaciens ; Roche) were used for pretreatment, as indicated in the figure legends.

Techniques: Blocking Assay, Injection, Isolation, Staining, Flow Cytometry

Journal: Cellular and Molecular Immunology

Article Title: Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2

doi: 10.1038/s41423-024-01142-0

Figure Lengend Snippet: Targeting sialoglycans and Siglec-9 on suppressive human CD33 + cells attenuates their function. A Experimental setup for generating suppressive myeloid cells in vitro. Fresh PBMCs were isolated from buffy coats of healthy donors and cocultured with the indicated cancer cell lines at a ratio of 100:1. On Day 7, CD33 + cells were isolated by magnetic positive selection, and their suppressive effect on autologous CD8 + T cells was assessed. Suppressive CD33 + cells were immediately used or were pretreated with sialidase or a Siglec-9 blocking antibody as indicated. CD8 + T cells were stained with CellTrace Violet (CTV) and stimulated by the addition of IL-2 and anti-CD3/28 microbeads. After 5 days, CD8 + T-cell proliferation was assessed by FACS. B Percentage of proliferating CD8 + cells upon coculture with the indicated suppressive CD33 + cells. Suppressive myeloid cells were generated from A459, A549 cells stably expressing sialidase (A549-sia) or A549 GNE KO cancer cells. N = 4–24 donors. A representative histogram for each condition is shown on the right. C Lectin staining was performed on suppressive CD33 + cells on Day 7 of the experiment to assess SNA and ( D ) MALII levels. Representative images of A549 (green) and A549-sia-MDSC-like cells (pink) are shown on the right. n = 7–10 donors in paired conditions . E Percentage of proliferating CD8 + cells upon coculture with suppressive CD33 + cells generated by A549 coculture. CD33 + cells were used immediately or were pretreated with sialidase or a Siglec-9 blocking antibody as indicated. N = 6–24 donors . F Assay setup to test the suppressive capacity of tumor-digested CD33 + cells. CD33 + cells were freshly isolated from the tumor homogenates of lung or colon cancer patients and used immediately or were pretreated with sialidase or a Siglec-9 blocking antibody. CD8 + cells were isolated from fresh PBMCs from the same donor and stained with CTV. The suppressive activity was assessed on Day 5 by flow cytometry. G Percentage of proliferating CD8 + cells of more than 3 generations upon coculture with tumor-derived CD33 + cells. MDSCs were left untreated and pretreated with sialidase or a Siglec-9 blocking antibody. Exemplary results for each condition are shown on the right. n = 5–8 per condition . The data are presented as the mean ± SD. A two-tailed paired t test was used. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: Additionally, viral sialidase (active H1N1 ; Sino Biological) and bacterial sialidase ( Arthrobacter ureafaciens ; Roche) were used for pretreatment, as indicated in the figure legends.

Techniques: In Vitro, Isolation, Selection, Blocking Assay, Staining, Generated, Stable Transfection, Expressing, Activity Assay, Flow Cytometry, Derivative Assay, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2

doi: 10.1038/s41423-024-01142-0

Figure Lengend Snippet: CCL2 is involved in T-cell suppression via the Siglec-sialoglycan axis in suppressive myeloid cells. A MCP-1/CCL2 found in the supernatant of murine MDSC:T-cell cocultures at the endpoint of the experiment from Fig. . MDSCs were untreated, or were pretreated with sialidase or a Siglec-E blocking antibody . n = 3 donors per group . B Correlation of MCP-1/CCL2 levels measured in supernatants of murine MDSC:T-cell cocultures at the endpoint and percentage of proliferating CD8 + T cells from the same conditions from ( A ). n = 3 donors per group . C Experimental setup: Neutralization of CCL2 using a neutralization antibody in SigE ΔLysM mice and SigE WT littermates bearing B16F10 tumors. Mice were injected with a CCL2 neutralization antibody up to 3 times a week (gray arrow) starting 1 day after subcutaneous B16F10 tumor injection (black arrow). Tumor growth and survival were monitored, and the suppressive capacity of MDSCs was analyzed in vitro. D Kaplan‒Meier survival curves from pooled data from 2 independent experiments. n = 5–8 mice per group . E B16F10 tumors at the endpoint ( C ) were digested, cocultured with pervanadate and analyzed by phospho-flow cytometry for phosphorylated STAT3 (pSTAT3). The percentage of pSTAT3 gated on total MDSCs is shown. Representative histograms for each condition are shown on the right. n = 3 mice per group . F Suppressive capacity of MDSCs against naïve T cells. Percentage of proliferating CD8 + T cells cocultured without MDSCs, ( G ) with MDSCs from SigE WT mice or ( H ) MDSCs from SigE ΔLysM mice with or without addition of CCL2 blocking antibody. N = 3–5 mice from N = 3 experiments . I Cytokine expression found in the supernatant of human primary CD33 + :CD8 + cell cocultures at the endpoint of the experiment from Fig. . CD33 + cells were left untreated or pretreated with sialidase. Z scores were calculated for each cytokine and are shown on a color scale from blue (low) to red (high). n = 3 donors per group . The data are presented as mean ± SD. Two-tailed paired t tests or unpaired t tests ( E ) were used. R shows the Pearson correlation coefficient. For survival analysis, the log-rank test was used, followed by the Šidák correction for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: Additionally, viral sialidase (active H1N1 ; Sino Biological) and bacterial sialidase ( Arthrobacter ureafaciens ; Roche) were used for pretreatment, as indicated in the figure legends.

Techniques: Blocking Assay, Neutralization, Injection, In Vitro, Flow Cytometry, Expressing, Two Tailed Test

Journal: Infection and Immunity

Article Title: A pleiotropic role of sialidase in the pathogenicity of Porphyromonas gingivalis

doi: 10.1128/iai.00344-23

Figure Lengend Snippet: Constructions and validations of P. gingivalis sialidase-deficient mutants and their isogenic complemented strains. (A) Complementation of Δ0352, a previously constructed PG0352 deletion mutant in W83, using pG108 shuttle vector. Primers P1/P2 were used to amplify PG0352 and its upstream promoter sequence (P); Primers P5/P6 were used to detect the tetQ cassette in pG108. (B) RT-PCR (top) and filter paper spot test (bottom) showed that the expression of PG0352 and sialidase activity were abolished in the Δ0352 mutant and restored in its isogenic complemented strain CΔ0352. (C) Schematic illustration of replacing PGN1608 with ermF in 33277 strain. PGN1608::ermF was constructed by two-step PCR with three pairs of primers (P9 to P14) as labeled, which was electroporated into 33277 to in-frame replace PGN1608 with ermF via DNA allelic exchange, generating the Δ1608 mutant. (D) RT-PCR and (E) filter paper spot test showed that the expression of PGN1608 and sialidase activity were abolished in Δ1608 and restored in its isogenic complemented strain CΔ1608 which was constructed using pG108 shuttle vector. Recombinant PG0352 (rPG0352) and substrate alone were used as positive and negative controls, respectively.

Article Snippet: Sialidase activity was detected by measuring fluorescence (λ ex = 350 nM; λ em = 450 nM) using a ChemiDoc Imaging System (Bio-Rad).

Techniques: Construct, Mutagenesis, Plasmid Preparation, Sequencing, Reverse Transcription Polymerase Chain Reaction, Spot Test, Expressing, Activity Assay, Labeling, Recombinant