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: Frontiers in Oncology

Article Title: ARID1A Downregulation Predicts High PD-L1 Expression and Worse Clinical Outcome in Patients With Gallbladder Cancer

doi: 10.3389/fonc.2022.787897

Figure Lengend Snippet: Bioinformatics analysis revealed the regulation of immune-related genes and signaling pathways in AT-rich interactive domain 1A (ARID1A)-inactivated tumors. (A) Heatmap of cytotoxic T lymphocyte (CTL) signatures in tumors with high or low ARID1A expression. (B) Volcano plot presenting the downregulation of genes involved in CD8+ T-cell activation in ARID1A-inactivated tumors. (C) Gene set enrichment analysis revealed the downregulation of the immune-related pathway in ARID1A-inactivated tumors.

Article Snippet: Tissue microarray (TMA) was established in this study using formalin-fixed, paraffin-embedded surgical specimens, and the specimens were stained immunohistochemically using appropriate antibodies (anti-ARID1A [1:600, ab182560, Abcam, USA]; anti-PD-L1 [1:200, SP142, Roche, Switzerland]; anti-PD1 [1:100, ab52587, Abcam]; anti-CD8 [1:400, ab4055, Abcam]).

Techniques: Expressing, Activation Assay

Journal: Discover Oncology

Article Title: Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases

doi: 10.1007/s12672-024-01056-3

Figure Lengend Snippet: Gene expression changes after IL-11Rα knockdown or IL-11 exogenous expression in the OS cells. a , Four shRNA sequences (G2, C1, H8, and H2) targeting IL-11Rα were stably expressed in KRIB cells. Gene expression was measured by microarray. Processed and log2-transformed gene expression data were compared to that of control cells. The heat map shows subtracted values for the gene probes in which values for shRNA sequences C1, H8, and H2 differed on average by at least twofold from those in the control cells. The color bar shows the fold-change difference from the values in the control cells. b , Quantitative reverse-transcription PCR analysis of the mRNA expression levels of the PRC2 complex members EZH2 , EED , and SUZ12 in SJSA1 (upper panel) and KRIB cells (lower panel). P—parental cells, E—empty vector, C1—IL-11Rα knockdown via shRNA C1, H8—IL-11Rα knockdown via shRNA H8, RQ—relative quantity. c Immunoblot of EZH2 and EED protein levels in SJSA1 and KRIB cells. β-actin served as a loading control. P—parental cells, E—empty vector, C1—IL-11Rα knockdown via shRNA C1, H8—IL-11Rα knockdown via shRNA H8, RQ—relative quantity.

Article Snippet: Expression of EZH2 was evaluated using a rabbit anti-EZH2 antibody (clone AC22; Cell Signaling Technology) diluted at 1:15 (vol/vol) and incubated for 45 min, followed by development using an LSAB + Kit (Dako).

Techniques: Gene Expression, Knockdown, Expressing, shRNA, Stable Transfection, Microarray, Transformation Assay, Control, Reverse Transcription, Plasmid Preparation, Western Blot

Journal: Discover Oncology

Article Title: Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases

doi: 10.1007/s12672-024-01056-3

Figure Lengend Snippet: Exogenous addition of human IL-11 to OS cells enhances PRC2 activity. Cells were treated with human recombinant IL-11 (hIL-11; 100, 200, and 300 ng) for 1 h and analyzed by a , qPCR for EZH2 , EED , and SUZ12 values, normalized to PPIA, and b , immunoblotting for H3K27 methylation status and expression of PRC2 complex member proteins (EZH2, SUZ12, and EED). Total histone and β-actin were used as loading controls. c Densitometry measurements of H3K27me3 levels normalized to total histone H3 for each dose of hIL-11. UT – untreated, **** P < 0.0001. d Kaplan–Meier curves showing probability of recurrence-free survival by extent of EZH2 staining. e – g The extent of positive EZH2 staining (percentage of positively stained nuclei) was scored on TMAs composed of 200 formalin-fixed, paraffin-embedded, decalcified human OS samples (141 primary and 59 metastatic). e Strong diffuse nuclear labeling. f Weak nuclear labeling. g Negative. Magnification 200X

Article Snippet: Expression of EZH2 was evaluated using a rabbit anti-EZH2 antibody (clone AC22; Cell Signaling Technology) diluted at 1:15 (vol/vol) and incubated for 45 min, followed by development using an LSAB + Kit (Dako).

Techniques: Activity Assay, Recombinant, Western Blot, Methylation, Expressing, Staining, Formalin-fixed Paraffin-Embedded, Labeling

Journal: Discover Oncology

Article Title: Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases

doi: 10.1007/s12672-024-01056-3

Figure Lengend Snippet: IL-11 activates EZH2 OS cells a Immunoprecipitation assay of EZH2 in CCH-OS-D and SJSA1 cells treated with human IL11 (200 or 400 ng) for 24 h. b CCH-OS-D cells were treated with human IL-11 for 48 h and then used for immunoblot analysis of phosphorylated EZH2-Y244. β-actin was used as loading control

Article Snippet: Expression of EZH2 was evaluated using a rabbit anti-EZH2 antibody (clone AC22; Cell Signaling Technology) diluted at 1:15 (vol/vol) and incubated for 45 min, followed by development using an LSAB + Kit (Dako).

Techniques: Immunoprecipitation, Western Blot, Control

Journal: Discover Oncology

Article Title: Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases

doi: 10.1007/s12672-024-01056-3

Figure Lengend Snippet: IL-11 mediates H3K27 trimethylation. a KRIB and SJSA1, EZH2shRNA and vector control cells were treated with human IL-11 for 48 h, Cells were harvested and used for immunoblot with H3K27 antibody. Total H3 was used as a control. b Immunoblot analysis of EZH2 knockdown and vector control in KRIB and SJSA1 cells. β-actin was used as a loading control

Article Snippet: Expression of EZH2 was evaluated using a rabbit anti-EZH2 antibody (clone AC22; Cell Signaling Technology) diluted at 1:15 (vol/vol) and incubated for 45 min, followed by development using an LSAB + Kit (Dako).

Techniques: Plasmid Preparation, Control, Western Blot, Knockdown

Journal: Discover Oncology

Article Title: Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases

doi: 10.1007/s12672-024-01056-3

Figure Lengend Snippet: Inhibition of OS lung metastases by GSK126 in vivo. a Luciferase imaging of mice injected with SJSA1 cells comparing day 3, when primary tumors had developed, and day 21, after primary tumors had been removed by amputation but before treatment initiation. The bottom panels shows luciferase imaging and lung tumors for mice treated with control-, vehicle (cyclodextrin)-, and EZH2 inhibitor (EZH2i; GSK126) by day 48 after the start of the regimen. b Quantification of luciferase activity. c number of tumor nodules in control-treated versus GSK126-treated mice. d Survival curves for all mice. e Weight of lungs in control- and GSK126-treated mice. F Representative images of H&E staining of explanted lungs treated with Vehicle and GSK126. Scale bar: 100 μm

Article Snippet: Expression of EZH2 was evaluated using a rabbit anti-EZH2 antibody (clone AC22; Cell Signaling Technology) diluted at 1:15 (vol/vol) and incubated for 45 min, followed by development using an LSAB + Kit (Dako).

Techniques: Inhibition, In Vivo, Luciferase, Imaging, Injection, Control, Activity Assay, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Spatial transcriptomics reveals prognostically LYZ + fibroblasts and colocalization with FN1 + macrophages in diffuse large B-cell lymphoma

doi: 10.1007/s00262-025-03968-7

Figure Lengend Snippet: Prognostic value of LYZ and LILRB4 proteins in DLBCL receiving R-CHOP ( n = 37, 10X) and aNSCLC receiving ICIs ( n = 29, 2X and 10X). A Representative IHCs staining of LYZ in patient 1 (PFS = 1697 days) and patient 2 (PFS = 341 days), LILRB4 in patient 3 (PFS = 64 days) and patient 4 (PFS = 826 days). B Kaplan–Meier survival curves of PFS grouped by the LYZ and LILRB4 expression in DLBCL. C Representative mIF staining of LYZ, LILRB4, and pan Cytokeratin. D Kaplan–Meier survival curves of OS grouped by the LYZ and LILRB4 expression in aNSCLC. E Kaplan–Meier survival curves of PFS grouped by the COL3A1 expression and FN1 + macrophages in DLBCL. DLBCL: diffuse large B cell lymphoma; R-CHOP: rituximab, cyclophosphamide, doxorubicin: vincristine, and prednisone; aNSCLC: advanced non-small cell lung cancer; ICIs: immune checkpoint inhibitors; IHC: immunohistochemistry; PFS: progression-free survival; mIF: multiple immunofluorescence; and OS: overall survival

Article Snippet: Rabbit anti-human COL3A1 IgG antibody , #63034S , Cell Signaling Technology.

Techniques: Staining, Expressing, Immunohistochemistry, Immunofluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Spatial transcriptomics reveals prognostically LYZ + fibroblasts and colocalization with FN1 + macrophages in diffuse large B-cell lymphoma

doi: 10.1007/s00262-025-03968-7

Figure Lengend Snippet: Prognostic significance of autoantibodies against COL1A2, COL3A1, and FN1 in patients with DLBCL receiving R-CHOP ( n = 20 and 125) and NSCLC treated with ICIs ( n = 36). A Volcano plot, boxplot, and representative density plots of patients illustrating the distribution of COL1A2 autoantibodies in DLBCL. B Kaplan–Meier curve for PFS based on COL1A2 autoantibody levels in DLBCL. C Kaplan–Meier curves for OS and PFS in NSCLC patients receiving immunotherapy, stratified by COL1A2 and COL3A1 mRNA levels in GSE128989 and autoantibody presence, alongside representative density plots of COL1A2 and COL3A1 autoantibodies. D Comparison of FN1 autoantibody levels between healthy controls and DLBCL, with Kaplan–Meier analysis for PFS based on FN1 autoantibody levels. DLBCL: diffuse large B cell lymphoma; R-CHOP: rituximab, cyclophosphamide, doxorubicin: vincristine, and prednisone; NSCLC: non-small cell lung cancer; ICIs: immune checkpoint inhibitor; OS: overall survival; and PFS: progression-free survival

Article Snippet: Rabbit anti-human COL3A1 IgG antibody , #63034S , Cell Signaling Technology.

Techniques: Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Spatial transcriptomics reveals prognostically LYZ + fibroblasts and colocalization with FN1 + macrophages in diffuse large B-cell lymphoma

doi: 10.1007/s00262-025-03968-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-human COL3A1 IgG antibody , #63034S , Cell Signaling Technology.

Techniques: Formalin-fixed Paraffin-Embedded, Gene Expression, RNA Sequencing, Sequencing, Biomarker Discovery, Clinical Proteomics, Microarray, Control, Immunohistochemistry, Microscopy, Immunofluorescence, Software