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: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Protective Role of DDIT4 in Helicobacter pylori -induced Gastric Metaplasia Through Metabolic Regulation of Ferroptosis

doi: 10.1016/j.jcmgh.2024.101448

Figure Lengend Snippet: Verification of DDIT4 expression and H pylori colonization in the gastric tissues of Ddit4 +/+ and Ddit4 -/- mice. ( A ) The qRT-PCR analysis indicating the mRNA levels of DDIT4 in the stomach tissues of Ddit4 +/+ and Ddit4 -/- mice. ( B ) The ratio of p-mTOR to total mTOR protein levels in infected WT and Ddit4-knockout mice. ( C ) Silver staining showing H pylori colonization in the gastric tissues of mice (Scale bar, 10 μm). ( D ) Colony-forming units (CFUs) per gram stomach in animals were evaluated for H pylori colonization by culture at 4 days after sacrifice. ( E ) IHC scores of ATP4A and ATP4B in infected gastric tissues of Ddit4 +/+ and Ddit4 -/- mice. ( F ) Immunofluorescence staining for MUC2 ( left panel ) or FABP1 ( right panel ) on infected gastric tissues from Ddit4 +/+ and Ddit4 -/- mice (Scale bar, 20 μm). ns, no significance. ∗ P < .05; ∗∗ P < .01; and ∗∗∗ P < .001 were considered significant.

Article Snippet: p-mTOR , Cell Signaling Technology , #5566 , WB , 1: 1000.

Techniques: Expressing, Quantitative RT-PCR, Infection, Knock-Out, Silver Staining, Immunofluorescence, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Protective Role of DDIT4 in Helicobacter pylori -induced Gastric Metaplasia Through Metabolic Regulation of Ferroptosis

doi: 10.1016/j.jcmgh.2024.101448

Figure Lengend Snippet: Ddit4 deletion results in gastric metaplasia in animal model of H pylori pathogenesis. ( A ) Western blot analysis verifying the loss of DDIT4 and activation of p-mTOR in gastric tissues of Ddit4 -/- mice. ( B ) Histopathology of gastric tissues of Ddit4 +/+ (n = 8) and Ddit4 -/- (n = 8) mice with or without H pylori infection (Scale bar, 10 μm). ( C ) Gastric pathology scores from the mice in panel B. ( D ) PAS staining showing prominent mucous cell metaplasia in gastric tissues (Scale bar, 10 μm). ( E ) Immunofluorescence staining for the expression of Ki67 in gastric tissues of WT ( Ddit4 +/+ ) and Ddit4 -/- mice with or without H pylori infection (Scale bar, 10 μm). ( F ) IHC staining for ATP4A and ATP4B expression in the gastric tissues of H pylori -infected Ddit4 +/+ and Ddit4 -/- mice (Scale bar, 10 μm). ( G ) The Western blot analysis showing the protein levels of ATP4A and ATP4B in gastric tissues of infected Ddit4 +/+ and Ddit4 -/- mice. ( H ) Immunofluorescence staining for GIF and GSII on the gastric tissues from H pylori -infected Ddit4 +/+ and Ddit4 -/- mice (Scale bar, 50 μm). ( I ) Immunofluorescence staining for CD44V9 on gastric tissues of Ddit4 +/+ and Ddit4 -/- mice infected with H pylori. (Scale bar, 20 μm). ∗P < .05; ∗∗ P < .01; and ∗∗∗ P < .001 were considered significant.

Article Snippet: p-mTOR , Cell Signaling Technology , #5566 , WB , 1: 1000.

Techniques: Animal Model, Western Blot, Activation Assay, Histopathology, Infection, Staining, Immunofluorescence, Expressing, Immunohistochemistry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Protective Role of DDIT4 in Helicobacter pylori -induced Gastric Metaplasia Through Metabolic Regulation of Ferroptosis

doi: 10.1016/j.jcmgh.2024.101448

Figure Lengend Snippet: Clinical relevance of DDIT4 and ferroptosis in human gastric intestinal metaplasia tissues. ( A ) Western blot data for the expression levels of DDIT4, ALOX15, HMOX1 and GPX4 in human GIM and non-atrophic gastritis (GS) tissues. ( B–C ) IHC staining showing DDIT4 and GPX4 expression in human GS and GIM tissues. Representative images (Scale bar, 10 μm) shown in ( B ). Quantification of staining shown in ( C ). ( D–E ) C11-BODIPY and DCFH-DA staining measured with a fluorescence microscope showing lipid peroxidation ( D ) and ROS levels ( E ) in human GS and GIM tissues. ( F ) Diagram of the mechanism by which loss of DDIT4 promoted H pylori -induced gastric metaplasia lesions through metabolic regulation of ferroptosis. Chronic H pylori infection causes the loss of DDIT4 and mTOR signaling pathway activation, which in turn inhibits GSH synthesis and increases ROS levels and lipid peroxides, inducing ferroptosis in the gastric microenvironment. As a result, gastric metaplasia lesions develop following parietal cell loss. ∗ P < .05; ∗∗ P < .01; and ∗∗∗ P < .001 were considered significant.

Article Snippet: p-mTOR , Cell Signaling Technology , #5566 , WB , 1: 1000.

Techniques: Western Blot, Expressing, Immunohistochemistry, Staining, Fluorescence, Microscopy, Infection, Activation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Protective Role of DDIT4 in Helicobacter pylori -induced Gastric Metaplasia Through Metabolic Regulation of Ferroptosis

doi: 10.1016/j.jcmgh.2024.101448

Figure Lengend Snippet: The Primary Antibodies Used in This Study

Article Snippet: p-mTOR , Cell Signaling Technology , #5566 , WB , 1: 1000.

Techniques: Immunohistochemistry-IF

Journal: Scientific Reports

Article Title: Regulation of folate and methionine metabolism by multisite phosphorylation of human methylenetetrahydrofolate reductase

doi: 10.1038/s41598-019-40950-7

Figure Lengend Snippet: Evidence for subsequent involvement of GSK3 in MTHFR phosphorylation. ( a ) Western blot analysis of immunoprecipitated, N-terminal-Flag-tagged wild-type MTHFR overexpressed from MDA-MB-468, 293FT, MCF7, and HelaS3, treated with 1 μM the GSK3 inhibitor, CHIR99021 for 0, 10 min, 60 min, and 3 h. P-GS denotes phospho-glycogen synthase Ser641, a known site for GSK3 . ( b ) The same as ( a ), except that a C-terminal Flag tag was knocked into both alleles of the endogenous MTHFR locus using CRISPR. ( c ) Western blot analysis of MDA-MB-468 parental or GSK3B -knockout cells, with or without doxycycline-inducible knockdown of GSK3A. DOX concentration was 1 μg/ml.

Article Snippet: SpTP motif, DYRK1A, GSK3A/B, GSK3A, P-glycogen synthase antibodies was purchased from Cell Signaling Technology (Cat# 5243, Cat# 2771, Cat#5676, Cat#4818, Cat#94905 respectively).

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, FLAG-tag, CRISPR, Knock-Out, Knockdown, Concentration Assay