Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1

doi: 10.1016/j.mcpro.2021.100160

Figure Lengend Snippet: Transcriptomic analysis identifies ST6GAL1 and ST3GAL3 as enriched in the cancerous ducts. A , biosynthetic pathways for glycans underlying select lectin signatures are shown. ST3GALs are responsible for transferring α-2,3-sialosides and ST6GAL1/2 for α-2,6-sialosides, and MGAT3 for bisecting GlcNAc. The glycans are annotated following the Symbolic Nomenclature for Glycans. B , transcriptomic analysis assessing the mRNA levels of select glycosyltransferases between normal adjacent and matched cancerous tissues within the same patient. C , uMAP plots representing the cells isolated from patients with PDAC (n = 24) and normal pancreata (n = 11) pooled on single-cell sequencing. The clusters representing the normal ductal cells and tumor ductal cells are highlighted. D , violin plot showing the comparison of ST6Gal1 levels in normal ( green ) versus PDAC ( blue ) ductal clusters. The clusters in each group are combined. The violin plots showing the comparison of ST6Gal1 and ST3GAL1 levels in normal ( green ) versus PDAC ( blue ) ductal clusters. The clusters in each group are combined. ns, p > 0.05; ∗ p < 0.05; student’s t test ( two-tailed ). MGAT3, beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase; ns, not statistical; PDAC, pancreatic ductal adenocarcinoma; ST3GAL3, ST3 beta-galactoside alpha-2,3-sialyltransferase 3; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1; uMAP, Uniform Manifold Approximation and Projection.

Article Snippet: The slide was then incubated with anti-human ST6GAL1 (Proteintech, cat # 14355-1-AP) followed by Opal Polymer horseradish peroxidase Ms + Rb (Akoya Biosciences, cat # ARH1001EA) and tyramide-linked 650 Opal fluorophore (Akoya Biosciences, cat # FP1496001KT) to amplify signals.

Techniques: Transferring, Isolation, Sequencing, Comparison, Two Tailed Test

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1

doi: 10.1016/j.mcpro.2021.100160

Figure Lengend Snippet: Profiling of ST6GAL1 and SNA in human pancreatic cancer shows association with the stage and survival. A , H&E of the normal pancreas ( left ), stage I pancreatic adenocarcinoma ( center ), and stage IV PDAC ( right ) stained from a BioMax human tissue microarray. B , multiplex OPAL IF staining of SNA ( yellow ), ST6GAL1 ( red ), and DAPI ( blue ) on the corresponding normal pancreas and stage I and stage IV pancreatic adenocarcinoma purchased from BioMax human tissue microarray. The scale bars represent 25 μm. C , quantification of SNA-positive cells per high-powered field based on multiplex IF in normal versus all cancerous cases in human tissue microarray. Owing to the high number of positive cells, each HPF was given a score (1–3) based on the number of SNA-positive cells per field. D , quantification of ST6GAL1-positive cells per high-powered field in normal versus all cancerous cases in human tissue microarray. E , distribution of the total ST6GAL1-positive cells per high-powered field across normal and each stage of pancreatic cancer based on multiplex IF imaging of human pancreatic cancer tissue microarray. Statistical significance was evaluated using the Student’s t test ( two-tailed ): ns, p > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. HFP, high-powered field; ns, not statistical; PDAC, pancreatic ductal adenocarcinoma; SNA, Sambucus nigra agglutinin; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1.

Article Snippet: The slide was then incubated with anti-human ST6GAL1 (Proteintech, cat # 14355-1-AP) followed by Opal Polymer horseradish peroxidase Ms + Rb (Akoya Biosciences, cat # ARH1001EA) and tyramide-linked 650 Opal fluorophore (Akoya Biosciences, cat # FP1496001KT) to amplify signals.

Techniques: Staining, Microarray, Multiplex Assay, Imaging, Two Tailed Test

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1

doi: 10.1016/j.mcpro.2021.100160

Figure Lengend Snippet: Pancreas-specific deletion of ST6GAL1 reduces disease burden in murine PDAC. A , breeding schematic illustrating the generation of novel ST6KC mice. The offspring of parental strains crossed into p48-Cre mice drive the induction of mutant KRAS G12D and deletion of ST6GAL1 under the same promoter. B , IHC staining for ST6GAL1 ( brown ) in KC and ST6KC mice (n = 3 per group). The number of ST6GAL1+ cells per high-powered field is quantified. The scale bar represents 50 μm. C , IF staining for SNA ( yellow ) and DAPI ( blue ) in KC and ST6KC mice (n = 3 per group). The number of SNA+ cells per high-powered field is quantified. The scale bar represents 100 μm. D , H&E of 14-week-old FFPE pancreata from KC and ST6KC mice (n = 5 per group). The percent of the preserved normal pancreas area is quantified per high-powered field. The scale bar represents 200 μm. E , trichrome and gomori ( blue ) stain of FFPE pancreata from 14-week-old KC and ST6KC mice (n = 5 per group). The percent of collagen deposition fibrosis is quantified per high-powered field on the right. The scale bar represents 200 μm. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; Student’s t test ( two-tailed ). FFPE, formalin-fixed paraffin-embedded; IF, immunofluorescence; IHC, immunohistochemical; PDAC, pancreatic ductal adenocarcinoma; SNA, Sambucus nigra agglutinin; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1; ST6KC, ST6GAL1 flx/flx ;p48 Cre ; LSL KRASG12D .

Article Snippet: The slide was then incubated with anti-human ST6GAL1 (Proteintech, cat # 14355-1-AP) followed by Opal Polymer horseradish peroxidase Ms + Rb (Akoya Biosciences, cat # ARH1001EA) and tyramide-linked 650 Opal fluorophore (Akoya Biosciences, cat # FP1496001KT) to amplify signals.

Techniques: Mutagenesis, Immunohistochemistry, Staining, Two Tailed Test, Formalin-fixed Paraffin-Embedded, Immunofluorescence, Immunohistochemical staining

Journal: Radiation Oncology (London, England)

Article Title: Cleavage of ST6Gal I by Radiation-Induced BACE1 Inhibits Golgi-Anchored ST6Gal I-Mediated Sialylation of Integrin β1 and Migration in Colon Cancer Cells

doi: 10.1186/1748-717X-7-47

Figure Lengend Snippet: Inhibition of BACE1 stabilizes ST6Gal I and increases cell surface sialylation . A. SW48 (ST6Gal I-negative) colon cancer cells were transiently transfected with an N-terminally Flag-tagged ST6Gal I expression plasmid, and 24 hours later, cells were exposed to IR (10 Gy). Cell lysates were assessed for expression of ST6Gal I by immunoblotting with an ST6Gal I antibody. B. SW480 vector-control cells and cells stably expressing ST6Gal I were treated with inhibitors of β-secretase, γ-secretase, or cathepsin B&L at a concentration of 20 μM. After incubating for 24 hours, cells were harvested and ST6Gal I levels were analyzed by immunoblotting with an anti-ST6Gal I antibody. C. Cell surface α2, 6 sialylation (SNA-FITC) and α2, 3 sialylation (MAA-FITC) were analyzed by FACS after 24 hours of treatment with 20 μM β-secretase or γ-secretase inhibitors. D. SW480 cells were transfected with siRNA against BACE1, and the levels of BACE1, ST6Gal I, and integrin β1 protein were assessed by immunoblotting. Sialylation of integrin β1 was assayed by SNA lectin affinity assay. E. SW480 vector-control cells and cells stably expressing ST6Gal I were treated with Si-BACE1 or transfected with a BACE1 expression plasmid. Then, α2, 6 sialylation (SNA-FITC) of the cell surface was analyzed by FACS. Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control).

Article Snippet: Protein levels were detected using the following commercial antibodies: anti-integrin β1 (BD Biosciences, Franklin Lakes, NJ, USA); anti-phospho-p130CAS, anti-phospho-Src, anti-HA, and anti-Myc (Cell Signaling Technology, Danvers, MA, USA); anti-Flag (Sigma); and anti-ST6Gal I and anti-BACE-1 (IBL, Japan).

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Western Blot, Stable Transfection, Concentration Assay

Journal: Radiation Oncology (London, England)

Article Title: Cleavage of ST6Gal I by Radiation-Induced BACE1 Inhibits Golgi-Anchored ST6Gal I-Mediated Sialylation of Integrin β1 and Migration in Colon Cancer Cells

doi: 10.1186/1748-717X-7-47

Figure Lengend Snippet: Sialylation and migration effects of non-cleavable ST6Gal I (L37A/K40A) . A. SW480 cells were transfected with WT ST6Gal I (WT) or ST6Gal I (L37A/K40A) and cell lysates were analyzed for the phosphorylated form of p130CAS (p-P130CAS Y410 ) and integrin β1 by immunoblotting. Expression of ST6Gal I was assessed using an anti-Flag antibody. Sialylation of integrin β1 was determined by SNA lectin affinity assay. B. SW480 cells were co-transfected with BACE1 and the WT or L37A/K40A form of ST6Gal I. Cell lysates were immunoblotted with antibodies against BACE1 and Flag. C. SW480 cells and CT26 cells were co-transfected with BACE1 and WT or L37A/K40A. Twenty-four hours after transfection, cell migration was determined using Transwell migration assays. Upper: images of migrated cells captured by phase-contrast microscopy; lower: migration measured by counting cells. Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control).

Article Snippet: Protein levels were detected using the following commercial antibodies: anti-integrin β1 (BD Biosciences, Franklin Lakes, NJ, USA); anti-phospho-p130CAS, anti-phospho-Src, anti-HA, and anti-Myc (Cell Signaling Technology, Danvers, MA, USA); anti-Flag (Sigma); and anti-ST6Gal I and anti-BACE-1 (IBL, Japan).

Techniques: Migration, Transfection, Western Blot, Expressing, Microscopy

Journal: Radiation Oncology (London, England)

Article Title: Cleavage of ST6Gal I by Radiation-Induced BACE1 Inhibits Golgi-Anchored ST6Gal I-Mediated Sialylation of Integrin β1 and Migration in Colon Cancer Cells

doi: 10.1186/1748-717X-7-47

Figure Lengend Snippet: The soluble form of ST6Gal I increases the migration of colon cancer cells . A. SW480 cells were transfected with WT ST6Gal I (WT) or ST6Gal I-ΔN (ΔN), and cell lysates were analyzed for the phosphorylated form of p130CAS (p-P130CAS Y410 ) and integrin β1 by immunoblotting. Sialylation of integrin β1 was determined by SNA lectin affinity assay. B. After transfection with the WT or ΔN form of ST6Gal I, cell migration was determined using Transwell migration assays. Upper: images of migrated cells captured by phase-contrast microscopy; lower: migration measured by counting cells. Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control).

Article Snippet: Protein levels were detected using the following commercial antibodies: anti-integrin β1 (BD Biosciences, Franklin Lakes, NJ, USA); anti-phospho-p130CAS, anti-phospho-Src, anti-HA, and anti-Myc (Cell Signaling Technology, Danvers, MA, USA); anti-Flag (Sigma); and anti-ST6Gal I and anti-BACE-1 (IBL, Japan).

Techniques: Migration, Transfection, Western Blot, Microscopy

Journal: FEBS letters

Article Title: Caveolin-1 up-regulates integrin α2,6-sialylation to promote integrin α5β1-dependent hepatocarcinoma cell adhesion.

doi: 10.1016/j.febslet.2013.02.002

Figure Lengend Snippet: Fig. 4. The increase in a2,6-sialylation on a5-subunit up-regulates FAK signaling. (A) The analysis of a2,6-linked sialic acid on a5-subunit from the total membrane protein of shNC, shCav-1-1, Cav-1-rescued and ST6Gal-I-rescued cells. The whole membrane lysates were immunoprecipitated (IP) with anti-a5 subunit antibody, and the blots were probed by SNA lectin (top) and anti-a5 subunit antibody (bottom) (WB, Western blot), respectively. IgG immunoprecipitates from shNC cells were used as a control. (B) Western blot analysis of a5- and b1-subunits protein levels in shNC, shCav-1-1, Cav-1-rescued and ST6Gal-I-rescued cells. (C) Flow cytometry analysis of a5- and b1-subunit levels on cell surface of shNC, shCav-1-1, Cav-1-rescued and ST6Gal-I-rescued cells. shNC cells stained only with FITC-conjugated IgG were as a control. (D) Western blot analysis of phosphorylated FAK, paxillin and ERK1/2 levels in shNC, shCav-1-1, Cav-1-rescued and ST6Gal-I-rescued cells treated with Fn (10 nM). Serum-starved cells were seeded into dishes coated with Fn (10 nM). The cell lysates were identified with either anti-p-FAK, anti-p-paxillin and anti-p-ERK1/2 antibodies to verify the phosphorylation levels of FAK, paxillin and ERK1/2 or with anti-FAK, anti-paxillin and anti-ERK1/2 antibodies to detect total amount of FAK, paxillin and ERK1/2.

Article Snippet: Antibodies against a5-subunit, b1-subunit, Cav-1, ST6Gal-I, FAK, phosphorylated FAK (p-FAK), paxillin, p-paxillin, ERK1/2, p-ERK1/2 and GAPDH (Santa Cruz Biotech Inc.) were used as the primary antibodies.

Techniques: Membrane, Immunoprecipitation, Western Blot, Control, Flow Cytometry, Staining, Phospho-proteomics

Journal: Redox Biology

Article Title: A Golgi-associated redox switch regulates catalytic activation and cooperative functioning of ST6Gal-I with B4GalT-I

doi: 10.1016/j.redox.2019.101182

Figure Lengend Snippet: Functional consequences of the inactivation of the ST6Gal-I. a) Immunoblotting of the ST6Gal and its ST6Gal-I CysDM variant in COS-7 cells. Cell transfections were performed by electroporation before SDS-PAGE and immunoblotting with ST6Gal-specific antibodies (anti-CD75) as above. b) Representative figures on SNA lectin binding to the cells transfected separately with the same plasmids. Transfections and lectin staining was carried out as describe above. c) Quantification of the SNA lectin binding to cells using the high content imaging system. The data are shown as relative changes (mean % + SD, n = 3, 70–90000 cells/assay). d) Live cell proliferation rate analysis of COS-7 cells transfected with an empty vector (EV) or with vectors expressing the wild-type ST6Gal-I (WT) or the cysteine double mutant of ST6Gal-I (CysDM). Data are expressed as mean confluence % (±SD (n = 5) using a build-in Incyte™ protocol. The star (*) mark the statistically significant difference (p < 0.05) between relative confluence values of the wild-type ST6Gal-I (WT) and the cysteine double mutant (ST6Gal-I CysDM ) at each time point. e) Live cell wound closure analysis of wild type (WT) and double cysteine (CysDM) ST6Gal-I variant expressing cells. The graph shows thee mean confluence (% ±SD (n = 4) at the indicated times after making the wound. Significant changes between confluence values between the wild-type ST6Gal-I (WT) and cysteine double mutant of ST6Gal-I (CysDM) at each time point are marked by stars (p < 0.05*). f) Representative images of the wound closure in COS-7 cells transfected with an empty vector (EV), ST6Gal-I (WT) or the cysteine double mutant of ST6Gal-I (CysDM) at 0 h, 60 h and 120 h time point after introduction of the wound. Scale bar 300 μm. Green color represents the original wound, and yellow color depicts the area of repopulated cells. g) Sensitivity cells to staurosporine-induced apoptosis. Cells transfected with the indicated ST6Gal-I variants were subjected to 5 μM staurosporine for 0–6 h. After fixing, cells were stained with the Hoechst dye before imaging high content imaging system (Operetta, PerkinElmer). Fragmentation index of nuclei was then calculated after image segmentation using the “RMS Nuclear Fragmentation” built-in protocol, in which the coefficient of variance percentage (CV%) of the nuclear intensity is used to calculate the fragmentation index. Healthy nuclei have low CV values whereas fragmented nuclei have higher CV values. The values represent the mean (±SD, n = 3, 15,000 cells each. Statistically significant changes are marked with stars (p < 0.05*, p < 0.01**, p < 0.001***). h-k) The effect of the wild type ST6Gal-I and its double cysteine variant on nuclear and cell morphology. Cells transfected with the indicated constructs were plated on a 96 well plate, fixed immuno-stained with an α-tubulin antibody, and counterstained with the Hoechst dye. After imaging (Operetta, PerkinElmer) and segmentation, in-built “Harmony” protocols were used to calculate nuclear and cytoplasmic areas (h and j) and their roundness (i and k). The data are expressed as the mean % (±SD, n = 3, 15,000 cells each) of the control values (EV = empty vector). No statistically significant changes were detected between the different ST6Gal-I transfectants (p > 0.05).

Article Snippet: For normalization, identical cell samples were subjected to SDS-PAGE and western blotting with the anti-CD75 antibody (#AP21150PU-N, Acris Antibodies GmbH, Germany) to determine the level of ST6Gal-I protein in each sample (Suppl.).

Techniques: Functional Assay, Western Blot, Variant Assay, Transfection, Electroporation, SDS Page, Binding Assay, Staining, Imaging, Plasmid Preparation, Expressing, Mutagenesis, Construct, Control