Journal: International Journal of Biological Sciences

Article Title: DNMT3a promotes LUAD cell proliferation and metastasis by activating the HDAC7 signalling pathway

doi: 10.7150/ijbs.96509

Figure Lengend Snippet: High expression of HDAC7 predicts poor prognosis in LUAD. a Kaplan-Meier survival analysis of patients represented in the Kaplan-Meier Plotter database stratified by HDAC7 expression. b Representative images of HDAC7 IHC staining in LUAD and adjacent nontumour tissues. Scale bars, 200 μm and 20 μm (inset). c Statistical analysis of HDAC7 expression based on IHC staining in tumour tissue and adjacent nontumour tissue of 119 LUAD patients. Statistical analysis of HDAC7 expression based on IHC staining of samples from 119 LUAD patients stratified into subgroups of T classification, N classification and clinical stage. Kaplan-Meier survival analysis of 119 LUAD patients based on HDAC7 expression. d Comprehensive Kaplan-Meier survival analysis of 119 LUAD patients stratified by DNMT3a and HDAC7 expression based on tissue microarray IHC results. e Correlation analysis of DNMT3a and HDAC7 expression in LUAD tissues. f Representative images and statistical analysis of the colony formation assay. Colonies were visualized by crystal violet staining. Representative images and statistical analysis of the EdU incorporation assay. The results were calculated as the ratio of the number of EdU-positive cells (red fluorescence) to the total number of Hoechst 33342-positive cells (blue fluorescence). Scale bar, 100 μm (inset). g Representative wound healing assay images and results. The migration ability was quantified as the mean scratch area at each time point. The initial scratch area (0 h) was set as 100%. Scale bars, 100 μm (inset). h Representative transwell migration assay images and results. Scale bars, 200 μm (inset). * p < 0.05. ** p < 0.01.

Article Snippet: According to standard practice, immunohistochemical (IHC) staining of paraffin-embedded tissue microarray sections was performed using primary antibodies of anti-DNMT3a (1:400, 20954-1-AP, Proteintech) and anti-HDAC7 (1:100, #33418, Cell Signaling Technology (CST)).

Techniques: Expressing, Immunohistochemistry, Microarray, Colony Assay, Staining, Fluorescence, Wound Healing Assay, Migration, Transwell Migration Assay

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: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Evaluation of pharmacodynamic responses to cancer therapeutic agents using DNA damage markers

doi: 10.1158/1078-0432.CCR-18-2523

Figure Lengend Snippet: (A) Baseline expression of selected DDR markers was demonstrated and quantified in a colorectal tissue array with 32 tumors (2 cores per case are included) and 15 normal colon tissues. At least 500 individual tumor nuclei were quantified in 95% of the tissue microarray cores. (B-C) Baseline marker quantitation in advanced stage colorectal cancers from patients enrolled in Phase 1 clinical trials at NCI. Median expression and inter-quantile range is indicated for each marker. A minimum of 4,000 individual tumor nuclei were quantitated across at least two nonadjacent slides per biopsy specimen. (D) Baseline expression in human colon adenocarcinoma patient-derived xenografts. At least 5,000 nuclei quantified per model. (E) Baseline expression of selected DDR markers was demonstrated and quantified in 8 colorectal cancer cell lines. Over 1000 individual nuclei were quantified for each cell line. (F) Inter-lesion baseline Rad51 quantitation from patients with advanced stage cancers with two biopsies each collected from the same lesion. *p< 0.05. The dashed line represents our empirically determined baseline value cutoff of 5% of cells ≥ 5 Rad51 foci per nucleus.

Article Snippet: Baseline biological variability was established for each biomarker across 64 individual cores from 32 colorectal (CRC) tumor resections contained in a paraffin-embedded colorectal tissue microarray (TMA; Indivumed, Hamburg, Germany).

Techniques: Expressing, Microarray, Marker, Quantitation Assay, Derivative Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Evaluation of pharmacodynamic responses to cancer therapeutic agents using DNA damage markers

doi: 10.1158/1078-0432.CCR-18-2523

Figure Lengend Snippet: Representative H&E and immunofluorescence (IFA) images from 3 patients with advanced colorectal cancer enrolled in NCI trial NCT01851369 before and 5 days after start of treatment with a DNA damaging therapeutic regimen consisting of TCR102 plus temozolomide administered orally once daily. Red scale bar represents 10 μm.

Article Snippet: Baseline biological variability was established for each biomarker across 64 individual cores from 32 colorectal (CRC) tumor resections contained in a paraffin-embedded colorectal tissue microarray (TMA; Indivumed, Hamburg, Germany).

Techniques: Immunofluorescence