Journal: Cancers

Article Title: In Silico and In Vitro Mapping of Receptor-Type Protein Tyrosine Phosphatase Receptor Type D in Health and Disease: Implications for Asprosin Signalling in Endometrial Cancer and Neuroblastoma

doi: 10.3390/cancers16030582

Figure Lengend Snippet: Log2 expression heatmaps generated from microarray experiments conducted on six healthy donors (data obtained from the Allen Brain Atlas). An analysis of the Allen Brain Atlas database indicated that Protein Tyrosine Phosphatase Receptor Type D (PTPRD) exhibits high expression levels in the occipital lobe, parietal lobe, globus pallidus, ventral thalamus, and white matter, and high to moderate expression within the hippocampal formation, hypothalamus, myelencephalon, and cerebral cortex regions of the brain (green arrows—moderate to high expression; orange arrows—predominantly high expression) .

Article Snippet: A paraffin-embedded tissue microarray (AMSBIO) was used for the present IHC work.

Techniques: Expressing, Generated, Microarray

Journal: Cancers

Article Title: In Silico and In Vitro Mapping of Receptor-Type Protein Tyrosine Phosphatase Receptor Type D in Health and Disease: Implications for Asprosin Signalling in Endometrial Cancer and Neuroblastoma

doi: 10.3390/cancers16030582

Figure Lengend Snippet: Protein Tyrosine Phosphatase Receptor Type D (PTPRD) expression in of a tissue microarray containing 43 endometrial cancer cores and 5 healthy tissue cores. Healthy ( panel a ), low-grade stage 1 ( panel b ), high-grade stage 1 ( panel c ), low-grade stage >1 ( panel d ), high-grade stage >1 ( panel e ), and positive control adrenal tissues ( panel f ). Immunohistochemical (IHC) scoring ± standard error of the mean (SEM) ( panel g ). Scale bar for images ( a – f ): 200 μM.

Article Snippet: A paraffin-embedded tissue microarray (AMSBIO) was used for the present IHC work.

Techniques: Expressing, Microarray, Positive Control, Immunohistochemistry

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

Journal: Current Issues in Molecular Biology

Article Title: Development of a Novel Anti-CD44 Variant 4 Monoclonal Antibody C 44 Mab-108 for Immunohistochemistry

doi: 10.3390/cimb45030121

Figure Lengend Snippet: Immunohistochemical analysis using C 44 Mab-108 and C 44 Mab-46 against oral squamous cell carcinoma (OSCC) tissues. After antigen retrieval, the sections were incubated with 10 µg/mL of C 44 Mab-108 ( A , B , I , J ), 1 µg/mL of C 44 Mab-46 ( C , D , K , L ), and without the primary antibody (control) ( E , F , M , N ) followed by treatment with the Envision+ kit. The color was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB), and the sections were counterstained with hematoxylin. ( G , H , O , P ) Hematoxylin and eosin (HE) staining. ( Q , R ) Blocking of the C 44 Mab-108 reactivity to OSCC tissue by the CD44 peptide (aa 271–290) containing the C 44 Mab-108 epitope. After antigen retrieval, sections were incubated with C 44 Mab-108 (10 μg/mL) or C 44 Mab-108 (10 μg/mL) plus human CD44 peptide (aa 271–290, 10 μg/mL) followed by treatment with the Envision+ kit. The color was developed using DAB, and sections were counterstained with hematoxylin. Scale bar = 100 µm.

Article Snippet: The formalin-fixed paraffin-embedded (FFPE) OSCC tissue microarray (Product Code: OR601c, US Biomax Inc., Rockville, MD, USA) and the esophageal tissue microarray (Product Code: BC02011, US Biomax Inc.) were deparaffinized in xylene (Sigma-Aldrich Corp.) and rehydrated.

Techniques: Immunohistochemical staining, Incubation, Staining, Blocking 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: 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: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) LGR5 IHC staining in normal human colon (one of five representative patients) at low (A1) and high (A2) magnification, as well as adenoma (A3) from the same patient (high-grade dysplasia; adjacent to adenocarcinoma; specimen 14881). (B) Lgr5 expression by in situ hybridization provides a conventional reference for the LGR5 IHC staining in normal crypts (upper panel) and in the adenoma (bottom panel); glandular Lgr5 (arrow-1) and stromal expression (arrow-2) in adenoma; (C) LGR5 IHC (C1, C2) and IF staining (C3, C4) in fetal duodenum, and (D) ISH expression in the same duodenum specimen. Scale bars, 100μM; A2, 25μM.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Immunohistochemistry, Expressing, In Situ Hybridization, Staining

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) LGR5 immunohistochemical and immunofluorescent staining in adult duodenum showed weak punctate LGR5 expression, while adjacent cells stained with the paneth cell marker DefensinA5 (DEFA5) (middle and right panel). Scale bars, 50μM. (B) Mouse 1881 lymphoma cells were previously transfected with human Lgr5 [1881(+); Miltenyi Biotec]. To confirm transfection stability, the 1881 LGR5(+) and 1881 Lgr5(−) cells were assayed for Lgr5 expression (n=3 biological replicates), and visualized with agarose gel analysis of qRT-PCR amplified products. Line represents mean values. (C) Measurement of human LGR5 protein expression by Western blotting with the rabbit monoclonal anti-human LGR5 antibody clone STE- 1-89-11.5 in 1881(+) cells, 282 adenoma organoids cultured in KGMG, and 14881 adenoma organoids cultured in KGMG or L-WRN.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Immunohistochemical staining, Staining, Expressing, Marker, Transfection, Agarose Gel Electrophoresis, Quantitative RT-PCR, Amplification, Western Blot, Cell Culture

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Representative images of LGR5 staining in the epithelial and stromal components in normal colon (n=5 normal autopsy samples) and from a CRC tissue microarray (n=2 normals, 65-68 neoplasm). Scale bar, 50μM. (B) Association between epithelial or (C) stromal staining intensity, and stage/grade of the tissue tested by generalized linear modeling, with staining intensity as the dependent variable and stage or grade as the independent variable.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Staining, Microarray

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Graphical representation of complete procedure. (B) Culture of adenoma organoids for LGR5 enrichment. LGR5 IHC staining (left column) in biopsied large adenoma (>10mm; two representative specimens are shown). Organoids with budding morphology (middle column) derived from these specimens cultured in the reduced medium KGMG. The same cultures with cystic morphology (right column) after being transferred for 3-4 weeks to L-WRN medium. Scale bars, 100μM. (C) Representative scatterplots of LGR5 expression in organoids cultured in KGMG or L-WRN (specimen 282). Live, LGR5(+) human adenoma cells were obtained after LGR5-magnetic bead enrichment by isolating DAPI(−) and LGR5(+) cellular populations.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Immunohistochemistry, Derivative Assay, Cell Culture, Expressing

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Relative LGR5 , SMOC2 , and DKK4 mRNA expression in L-WRN vs KGMG adenoma organoid cultures. Individual specimen qRT-PCR technical replicates; represents the mean±s.e.m. of n=3 biological replicates. (B) LGR5 immunohistochemistry staining and (C) SMOC2 and Lgr5 mRNA in situ hybridization (ISH) of formalin fixed paraffin-embedded (FFPE) adenoma 282 organoids cultured in L- WRN. Arrows designate localization of Lgr5 and SMOC2 in corresponding regions of consecutive serial sections. Scale bar, 50μM. (D) DKK4 and Lgr5 ISH images in consecutive FFPE cuts of a colon adenocarcinoma (specimen 815). Scale bar, 50μM.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, In Situ Hybridization, Formalin-fixed Paraffin-Embedded, Cell Culture

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Flow cytometric analysis for enhanced number and fluorescent intensity of LGR5(+) cells resulting from organoids cultured in the reduced medium KGMG and then transferred for 34 weeks to L-WRN medium. (B) Flow cytometry spike-in experiments to confirm LGR5 antibody specificity and to validate the magnetic bead enrichment strategy. (1) Pure populations of 1881 LGR5(+) and 1881 Lgr5(−) cells were analyzed by flow to establish gating strategy. 1881 LGR5(+) and 1881 Lgr5(−) cells were mixed in the proportions, (2) 50/50 and (3) 5/95; and analyzed by flow cytometry before (left column) and after magnetic separation (middle/right columns) with rat monoclonal anti-human LGR5 antibody clone 22H2.8.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Cell Culture, Flow Cytometry

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Multidimensional scaling plot of the LGR5(+) and LGR5(−) cells, based on the top 500 most variable genes. Patient identifiers #14881, 228, 584, and 590. (B) False discovery rate (FDR) volcano plot of the log(2) ratio of gene expression between the LGR5(+) and LGR5(−) cells. (C) Unsupervised hierarchical clustering heatmap of the 50 most differentially expressed genes by fold change between the LGR5(+) (red) and LGR5(−) (green) populations. (D) Log(2) fold change in gene expression between LGR5(+) and LGR5(−) cells for known markers of colon stem (red) and differentiated (green) cells. (E) The top 10 most enriched KEGG pathways for differentially expressed genes between the LGR5(+) and Lgr5(−) cells.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Multidimensional scaling plot of the LGR5(+), sorted MACS magnet-bound Lgr5(−) cells, and sorted MACS flowthrough LGR5(FT-) cells based on the top 500 most variable genes. (B) Unsupervised hierarchical clustering heatmap of the 50 most variable genes between the LGR5(+) (red), Lgr5(−) (green), and LGR5(FT-) (blue) populations. (C) The top 10 most enriched KEGG pathways for differentially expressed genes between the LGR5(+) and Lgr5(−); and LGR5(+) and LGR5(FT-) cells. (D) Gene expression overlap between the [LGR5(+) vs. Lgr5(−)], [LGR5(+) vs. LGR5(FT-)], and [Lgr5(−) vs. LGR5(FT-)].

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Concordance of adenoma LGR5(+) cell gene expression with gene expression associated with Wnt signaling in colon cancer stem cells, based on TOP-GFP Wnt reporter activity . (B) Brightfield and fluorescence images of the 282 adenoma organoid line lentivirally transduced with a TCF/LEF -GFP reporter for Wnt signaling. Scale bar, 50μM. (C) Representative ImageStream flow cytometry results of TCF/LEF -GFP organoids stained with the LGR5- APC antibody (n=3 experimental replicates). Scale bar, 10μM. (D) Comparison of LGR5 expression in the 10% highest and 10% lowest TCF/LEF -GFP expressing cells; values in iii and iv represent mean % ± s.e.m.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Gene Expression, Activity Assay, Fluorescence, Transduction, Flow Cytometry, Staining, Comparison, Expressing

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Gene expression overlap between genes upregulated in LGR5(+) vs. LGR5(−) cells and the Lgr5 mouse intestinal stem cell signature reported in Munoz et al . (B) Comparison of gene expression between LGR5(+) adenoma cells and previously published RNA-seq data from normal human colon for genes in the “human adenoma LGR5+ cell gene signature. (C) The top ten most differentially expressed genes by magnitude between LGR5(+) adenoma cells and normal human colon (all genes; unbiased analysis) . (D) An analysis of TCGA colorectal cancer gene expression data through Oncomine™ of DKK4, comparing colorectal adenocarcinoma expression with normal colon and rectum.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Gene Expression, Comparison, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Flow cytometry gating strategy for LGR5(+) cells isolated directly from warm autopsy normal colon tissue (specimen 84, a representative scatter plots of 3 patients; ). Includes EpCAM(+) inclusion criteria, using a phycoerythrin (PE)-conjugated antibody (not shown). To set the LGR5-APC(+) gating, either magnet flow-through (specimen 81 & 83), as done with organoid sorts, or rigorous so-called fluorescence- minus-one controls [FMO; DAPI(−), EpCAM(+); specimen 84] were used. (B) mRNA expression by qRT-PCR of Lgr5 and OLFM4 across all 3 patient samples. Values represent mean ± SE. (C) OLFM4 immunofluorescent staining of normal colon crypts. Scale bar, 100μM. (D) Normal colon organoid cultures were established from warm autopsy resections. LGR5(+)/(−) cells were isolated from early passage organoids, sorted into culture medium and seeded into Matrigel to access organoid-forming efficiency. Mean organoid-forming efficiency from two normal colon organoid cultures (specimen 78, passage 5; and specimen 85, passage 3) sorted for Lgr5(−) vs. LGR5(+) cells (n=4 wells per fraction), error bars represent s.e.m. Representative images (right panels; specimen 85) of organoids formed from Lgr5(−) and LGR5(+) cells shown at day 14 and day 52 (passage 5). Scale bars, 200μM top panel; 1mm bottom panel.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Flow Cytometry, Isolation, Fluorescence, Expressing, Quantitative RT-PCR, Staining

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: Relative expression of genes involved in the KEGG pathway “Glycolysis and Gluconeogenesis” between LGR5(+) and LGR5(−) human adenoma cells ( P -value=6.57E-09).

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Expressing