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: Cancers

Article Title: Tumor Expression of Cyclin-Dependent Kinase 5 (Cdk5) Is a Prognostic Biomarker and Predicts Outcome of Oxaliplatin-Treated Metastatic Colorectal Cancer Patients

doi: 10.3390/cancers11101540

Figure Lengend Snippet: Cyclin dependent kinase 5 (Cdk5) and p35 (CDK5R1) expression in colorectal cancer (CRC) cell lines. ( A ) Western blot analysis of Cdk5 and p35 basal expression in a panel of 10 CRC cell lines. Alpha-tubulin was used as endogenous control. ( B ) Representative western blot images showing the co-immunoprecipitation of Cdk5 and p35/p25 in the indicated cell lines. Co-immunoprecipitation with an immunoglobin G (IgG) antibody was used as a negative control. The results were obtained from at least three independent experiments.

Article Snippet: Cells were homogenized in lysis buffer and the supernatant was incubated with a Cdk5 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; (J3): sc-6247) overnight at 4 °C.

Techniques: Expressing, Western Blot, Control, Immunoprecipitation, Negative Control

Journal: Cancers

Article Title: Tumor Expression of Cyclin-Dependent Kinase 5 (Cdk5) Is a Prognostic Biomarker and Predicts Outcome of Oxaliplatin-Treated Metastatic Colorectal Cancer Patients

doi: 10.3390/cancers11101540

Figure Lengend Snippet: Effect of Cyclin dependent kinase 5 (Cdk5) siRNA-mediated gene silencing on colorectal cancer (CRC) cells proliferation, migration and invasion. ( A ) Graphic representation of HT29, LoVo, DiFi, and HCT116 time-dependent cell proliferation after Cdk5 gene silencing measured by propidium iodide (PI). ( B , D ) Representative Boyden chamber migration and invasion assays images (4× magnification) and ( C , E ) bar graphs showing (mean ± SD) relative cell migration and invasion after Cdk5 silencing in the indicated cell lines. * p -value < 0.05 relative to control cells siRNA non-target control (siNTC). The results were obtained from at least three independent experiments.

Article Snippet: Cells were homogenized in lysis buffer and the supernatant was incubated with a Cdk5 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; (J3): sc-6247) overnight at 4 °C.

Techniques: Migration, Control

Journal: Cancers

Article Title: Tumor Expression of Cyclin-Dependent Kinase 5 (Cdk5) Is a Prognostic Biomarker and Predicts Outcome of Oxaliplatin-Treated Metastatic Colorectal Cancer Patients

doi: 10.3390/cancers11101540

Figure Lengend Snippet: Overview of cohort used in the study.

Article Snippet: Cells were homogenized in lysis buffer and the supernatant was incubated with a Cdk5 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; (J3): sc-6247) overnight at 4 °C.

Techniques: In Silico, Microarray, Formalin-fixed Paraffin-Embedded, RNA Sequencing

Journal: Cancers

Article Title: Tumor Expression of Cyclin-Dependent Kinase 5 (Cdk5) Is a Prognostic Biomarker and Predicts Outcome of Oxaliplatin-Treated Metastatic Colorectal Cancer Patients

doi: 10.3390/cancers11101540

Figure Lengend Snippet: Cyclin dependent kinase (Cdk5) and p35 expression in colorectal cancer (CRC) tumor samples. Western blot ( A ) and graphic representation ( B , C ) of Cdk5 and p35 protein expression, respectively, in tumoral (T) and normal adjacent (N) tissues of 12 stage IV CRC patients (cohort A). Alpha-tubulin was used as endogenous control. The p -value was according to paired t -test. ( D ) Representative immunohistochemistry images of Cdk5 staining in CRC tumor tissues. The upper panel shows negative staining and lower panel positive staining. Scale bar: 100 μm. ( E ) Graphic representation of Cdk5 and p35 mRNA expression in 98-paired adjacent normal and tumoral tissues from stage II microsatellite stable (MSS) CRC patients (cohort B). ( F ) Graphic representation of Cdk5 mRNA expression in normal and tumoral tissues of 38 I-IV CRC patients. Data were obtained from The Cancer Genome Atlas (TCGA) database (cohort F). ( G ) Graph representing the Cdk5 copy number (CNV) change between normal and tumoral tissues in 67 stage I–IV CRC patients (cohort F). ( H ) Correlation between Cdk5 CNV and Cdk5 gene expression in 429 stage I–IV CRC patients. p-value according to Pearson correlation test (cohort F). ( I ) Correlation between Cdk5 CNV and Cdk5 gene expression in 63 CRC cell lines. The p -value was according to Pearson correlation test. Data from the Broad Institute Cancer Cell Line Encyclopedia.

Article Snippet: Cells were homogenized in lysis buffer and the supernatant was incubated with a Cdk5 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; (J3): sc-6247) overnight at 4 °C.

Techniques: Expressing, Western Blot, Control, Immunohistochemistry, Staining, Negative Staining, Gene Expression

Journal: Cancers

Article Title: Tumor Expression of Cyclin-Dependent Kinase 5 (Cdk5) Is a Prognostic Biomarker and Predicts Outcome of Oxaliplatin-Treated Metastatic Colorectal Cancer Patients

doi: 10.3390/cancers11101540

Figure Lengend Snippet: Kaplan–Meyer analysis of disease free survival (DFS) and time to progression (TTP) depending on Cyclin dependent kinase (Cdk5) levels. ( A ) DFS in 98 stage II colorectal cancer (CRC) patients split by the median of Cdk5 expression (cohort B). ( B ) DFS in 37 non-treated stage III CRC patients split by the median of Cdk5 expression (cohort C). ( C ) TTP in 52 stage IV oxaliplatin-treated patients, grouped depending on negative ( n = 18) or positive Cdk5 ( n = 34) immunohistochemistry (IHC) staining (cohort D). ( D ) TTP in 139 stage IV irinotecan-treated patients grouped depending on negative ( n = 73) and positive ( n = 66) Cdk5 IHC staining (cohort E).

Article Snippet: Cells were homogenized in lysis buffer and the supernatant was incubated with a Cdk5 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; (J3): sc-6247) overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry

Journal: Cancers

Article Title: Tumor Expression of Cyclin-Dependent Kinase 5 (Cdk5) Is a Prognostic Biomarker and Predicts Outcome of Oxaliplatin-Treated Metastatic Colorectal Cancer Patients

doi: 10.3390/cancers11101540

Figure Lengend Snippet: Kaplan–Meyer analysis of disease free survival (DFS) depending on Cyclin dependent kinase (Cdk5) expression and Kirsten rat sarcoma oncogene ( KRAS) mutational status. ( A ) DFS for 72 stage II colorectal cancer (CRC) patients with wildtype (WT) KRAS and split by the median of Cdk5 expression. ( B ) DFS for 26 stage II CRC patients with mutated KRAS and split by the median of Cdk5 expression (cohort B).

Article Snippet: Cells were homogenized in lysis buffer and the supernatant was incubated with a Cdk5 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; (J3): sc-6247) overnight at 4 °C.

Techniques: Expressing

Journal: Cancers

Article Title: Tumor Expression of Cyclin-Dependent Kinase 5 (Cdk5) Is a Prognostic Biomarker and Predicts Outcome of Oxaliplatin-Treated Metastatic Colorectal Cancer Patients

doi: 10.3390/cancers11101540

Figure Lengend Snippet: Cyclin dependent kinase 5 (Cdk5) expression in the four consensus molecular subtypes (CMS). ( A ) Dunn and Kruskal–Wallis multiple comparison of Cdk5 expression in CMS in 98 stage II tumors. Note that only six cases were classified as CMS1 as this cohort was restricted to microsatellite stabile (MSS) cases ( B ) ANOVA analysis of Cdk5 expression levels in the CMS in the cancer genome atlas - colorectal adenocarsinoma - rectal adenocarcinoma dataset (TCGA-COAD-READ) including 410 stage I–IV patients. * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001. ( C ) PFI in the TCGA-COAD CMS1 subgroup including 78 stage I–IV patients; patients were split according to the median of Cdk5 expression (cohort F).

Article Snippet: Cells were homogenized in lysis buffer and the supernatant was incubated with a Cdk5 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; (J3): sc-6247) overnight at 4 °C.

Techniques: Expressing, Comparison

Journal: Oncotarget

Article Title: Deficiency in p53 is required for doxorubicin induced transcriptional activation of NF-κB target genes in human breast cancer

doi:

Figure Lengend Snippet: Cells were cultured 90min in the presence or absence of MLN120B (20µM) and further incubated 4h with or without doxorubicin (5µM). Different steps of the NF-κB pathway were characterized. A, Levels of IκBα protein in whole protein lysates determined by Western Blot. α-tubulin served as the loading control. B, Representative images of p65 cellular distribution (nucleus/cytoplasm) determined by immunofuorescence (IF) using Alexa 488-coupled goat anti-rabbit IgG (green). Arrows indicate representative nuclear p65 signals in doxorubicin treated cells. C, DNA binding activities of NF-κB in nuclear extracts determined by EMSA. Lane 2 contains competitor “cold” NF-κB probe at 100-fold molar excess with the same nuclear extract than in Lane 1 as specificity control. D, DNA-binding activity of five NF-κB subunits analyzed in nuclear extracts using commercial ELISA kit. Bars represent the average of three independent experiments. Error bars represent standard deviations. E, MDA-MB-231 cells stably transfected with a NF-κB-luciferase reporter. To assay luciferase activity doxorubicin treatment was performed for 24h. Bars represent the average of three independent experiments. Error bars represent standard deviations. The results are reported as the percentage of fold increase in relative luminescence in arbitrary units (RLA) of treated sample using untreated condition as reference. F, Microarray gene expression profile was established in treated MDA-MB-231 cells. On the heat map, right panel indicates the genes differentially expressed comparing doxorubicin treated samples vs. samples treated with both MLN120B and doxorubicin. Green color indicates under expressed (down-regulated) genes; Red color indicates overexpressed (up regulated genes) and Black indicates no change.

Article Snippet: Western blot was performed as previously described [ ], using anti-IκBα, anti- ICAM-1 and anti-p53 antibodies (Cell Signaling, Danvers, MA), anti- NF-κB p65 and p50 (Santa Cruz Biotech, Dallas, TX) and anti-α-tubulin antibody (Sigma-Aldrich) as loading control.

Techniques: Cell Culture, Incubation, Western Blot, Control, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, Luciferase, Microarray, Gene Expression

Journal: Oncotarget

Article Title: Deficiency in p53 is required for doxorubicin induced transcriptional activation of NF-κB target genes in human breast cancer

doi:

Figure Lengend Snippet: Uni- and multivariate Cox analysis for disease-free survival in series of breast cancer patients (n=335), including clinico-patological factors and p65 and p53 53 nuclear expression

Article Snippet: Western blot was performed as previously described [ ], using anti-IκBα, anti- ICAM-1 and anti-p53 antibodies (Cell Signaling, Danvers, MA), anti- NF-κB p65 and p50 (Santa Cruz Biotech, Dallas, TX) and anti-α-tubulin antibody (Sigma-Aldrich) as loading control.

Techniques: Expressing, Adjuvant, Over Expression

Journal: Oncotarget

Article Title: Deficiency in p53 is required for doxorubicin induced transcriptional activation of NF-κB target genes in human breast cancer

doi:

Figure Lengend Snippet: A set of 20 human breast tumors were exposed ex vivo to vehicle, 20 µM MLN120B, 2 µg/ml doxorubicin or both during 24h and then formalin-fixed paraffin embedded (FFPE) tissues were prepared. A, Representative immunohistochemical images of FFPE sections control and doxorubicin treated stained with p65 and p50. B, Graph showing the IHC HScores for nuclear p65 and p50 stainining in all samples relative to control condition. C, Total RNA from ex vivo human breast tumors was extracted for analysis of the ICAM-1 and TNFAIP3 and CXCL-1 genes by qRT-PCR. The relative target gene expression level was also normalized to the RPLP0 expression in each sample. D, Representative immunohistochemical results of ICAM-1 staining in sections of the FFPE breast specimens; control (left panel), doxorubicin 5µM 24h (middle panel) and 20µM MLN120B plus doxorubicin 5µM 24 hours (right panel). E, Graph show the average of ICAM-1 expression determined by immunohistochemistry in all samples relative to control condition.

Article Snippet: Western blot was performed as previously described [ ], using anti-IκBα, anti- ICAM-1 and anti-p53 antibodies (Cell Signaling, Danvers, MA), anti- NF-κB p65 and p50 (Santa Cruz Biotech, Dallas, TX) and anti-α-tubulin antibody (Sigma-Aldrich) as loading control.

Techniques: Ex Vivo, Formalin-fixed Paraffin-Embedded, Immunohistochemical staining, Control, Staining, Quantitative RT-PCR, Targeted Gene Expression, Expressing, Immunohistochemistry

Journal: Oncotarget

Article Title: Deficiency in p53 is required for doxorubicin induced transcriptional activation of NF-κB target genes in human breast cancer

doi:

Figure Lengend Snippet: High-risk patients were stratified according to high or low p53 nuclear accumulation status and high or low nuclear NF-κB p65 staining.

Article Snippet: Western blot was performed as previously described [ ], using anti-IκBα, anti- ICAM-1 and anti-p53 antibodies (Cell Signaling, Danvers, MA), anti- NF-κB p65 and p50 (Santa Cruz Biotech, Dallas, TX) and anti-α-tubulin antibody (Sigma-Aldrich) as loading control.

Techniques: Staining