Journal: iScience

Article Title: Distinct but interchangeable subpopulations of colorectal cancer cells with different growth fates and drug sensitivity

doi: 10.1016/j.isci.2023.105962

Figure Lengend Snippet:

Article Snippet: For MSI1 overexpression, human MSI1 cDNA was amplified with the primers of MSI1_BamHI-F and MSI1_stopdead_XbaI-R ( ) using the PrimeScriptTM first strand cDNA Synthesis (Takara, 6210A) and PrimeSTAR Ⓡ Max DNA Polymerase kits according to manufacturer instructions (Takara, R045A).

Techniques: Recombinant, Concentration Assay, Membrane, RNAscope, Cell Viability Assay, SYBR Green Assay, Reverse Transcription, Expressing, Microarray, Derivative Assay, Software

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Stable loss of major histocompatibility complex class I (MHC-I) gene DNA methylation (DNAm) in intestinal epithelial organoids (IEOs) derived from patients with Crohn’s disease (CD). (A) (i) Overview of experimental set-up and sample generation. (ii) Representative brightfield images of IEOs. Scale bars: 300 µm. (B) (i) Correlation heat map of comethylated CpG modules identified by weighted gene coexpression network analysis (WGCNA) in terminal ileum (TI) IEOs. Module 17 (ME17) demonstrates hypomethylation and the strongest association with CD diagnosis (R=−0.43, p value<0.001). (ii) Gene set enrichment analysis performed on module 17, showing a significant loss of DNAm in CD organoids compared with healthy controls and UC in TI. (C) DNAm (beta value) of four representative MHC-I related Differetial Methylated Positions (DMPs) showing CD-associated loss of DNAm in TI and sigmoid colon (SC) but not duodenum (DUO) organoids (DUO=54, TI=127 and SC=131). (D) Average DNAm (beta value) of all CpGs located in MHC-I related genes for IEOs split by diagnosis, gut segment and inflammatory status. (E) (i) Correlation of nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) promoter DNAm between early and later passage IEOs from the same individuals including patients diagnosed with CD (blue), UC (yellow) and controls (grey, n=22 patients. (ii) DNAm (beta values) of CpGs located in NLRC5 and TAP1 at high passage (>7) IEOs (cohort 1, n=22). (F) Average MHC-I (i) and NLRC5 (ii) DNAm as well as NLRC5 gene expression (iii) in control patient-derived TI IEOs stimulated with proinflammatory cytokines interferon γ (IFNγ) and tumour necrosis factor α (TNFα) (n=5). (False Discovery Rate (FDR) * < 0.05, FDR **< 0.01, FDR***< 0.001, FDR**** < 0.0001, ns=not significant.)

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, DNA Methylation Assay, Derivative Assay, Biomarker Discovery, Methylation, Binding Assay, Gene Expression, Control

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Loss of major histocompatibility complex class I (MHC-I) DNA methylation (DNAm) correlates with increased gene expression in primary intestinal epithelium of patients with Crohn’s disease (CD). (A) Overview of patient cohort, sample preparation and data generation. (B, C) DNAm and gene expression in purified terminal ileum (TI) (B) and sigmoid colon (SC) (C) epithelium. (i) Average DNAm (beta value) of all and selected MHC-I pathway-related CpGs showing significant, CD-associated loss of DNAm. (ii) Correlation between beta values and corresponding gene expression (R=Spearman’s rank correlation). (D) Nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) promoter DNAm in the IE of healthy, patients with UC and CD at the point of diagnosis and during reassessment. (E) Correlation of NLRC5 promoter DNAm in intestinal epithelial organoids (IEOs) obtained from the same patient at diagnosis and reassessment (Spearman’s rank correlation). (F) NLRC5 promoter DNAm in TI IEOs derived from patients with CD, UC and control (n=3 IEO per condition, two-way analysis of variance (ANOVA) with Turkey’s test for multiple comparisons. ****p<0.0001). (G) NLRC5 mRNA expression in TI IEOs derived from patients with controls, UC and CD at baseline and on interferon γ (IFNγ) treatment (10 ng/mL for 6 hours). Data are normalised to the mean of control lines and shown as mean±SEM (two-way ANOVA with Turkey’s test for multiple comparisons. **P<0.01, *p<0.05, ns=not significant). n=3 IEO lines in each group for three independent experiments.

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, DNA Methylation Assay, Gene Expression, Sample Prep, Purification, Binding Assay, Biomarker Discovery, Derivative Assay, Control, Expressing

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) acts as transcriptional transactivator of intestinal epithelial cell (IEC) major histocompatibility complex class I (MHC-I) and potentiates the effect of interferon γ (IFNγ). (A) Overview of experimental set-up. (B) Heatmap showing gene expression (RNAseq) of MHC-I pathway genes in terminal ileum (TI) intestinal epithelial organoids (IEOs)± NLRC5 overexpression (dox), and ±exposure to IFNγ (n=4 independent replicates). (C) RNA transcription of HLA-A / -B / -C / -E / -F / -G in response to IFNγ and tumour necrosis factor α (TNFα) in wild type (WT) and NLRC5 OE TI IEOs. (D) Relative expression for MHC-I pathway genes in WT ( NLRC5 +/+ ) and corresponding NLRC5 deficient ( NLRC5 −/ − ) TI IEOs±IFNγ (n=3 replicates. Two-way analysis of variance (ANOVA) with Bonferroni’s test for multiple comparisons, **p<0.01, ***p<0.001, ****p<0.0001). Data are representative of two independent experiments. (E) Immunofluorescence spinning disc microscopy of organoids described in D, ±IFNγ (48 hours). (i) Representative images of untreated (BSA) and treated (IFNγ) WT ( NLRC5 +/+ ) and NLRC5 deficient ( NLRC5 −/ − ) TI IEOs taken by Opera Phoenix. Scale bar=2 mm. (ii) HLA-A,B,C mean intensity quantification of BSA and IFNγ NLRC5 +/+ and NLRC5 −/ − TI IEOs. (n=3 independent replicates. Two-way ANOVA with Bonferroni multiple comparisons test, **p<0.01, ***p<0.001, ****p<0.0001.) (F) Correlation between mRNA gene expression of NLRC5 and (i) HLA-B and (ii) HLA-E, in purified TI and sigmoid colon (SC) epithelium (cohort 2) (Spearman’s rank correlation).

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Binding Assay, Immunopeptidomics, Gene Expression, Over Expression, Expressing, Immunofluorescence, Microscopy, Purification

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Crohn’s disease (CD)-associated increased intestinal epithelial major histocompatibility complex class I (MHC-I) expression affects the stem cell compartment and follows a crypt-villus gradient. (A) Summary of experimental set-up. (B) (i) Schematic representation of intestinal epithelial cell (IEC) subtypes and their location within the small bowel (terminal ileum (TI)) crypt-villus structure (TA—transiently amplifying cells). (ii) Uniform manifold approximation and projection (UMAP) plot demonstrating single IEC transcriptomes present in TI mucosal biopsies obtained from children newly diagnosed with CD and non-IBD controls. (C) Top panel: violin plots showing crypt-villus scores of cells within each identified cell subtype (top left) and total number of cells (top right). Bottom panel: correlation between MHC-I summary score and crypt-villus scores for all IEC transcriptomes. Best fitting correlation is displayed as individual lines for CD (blue), UC (yellow) and non-IBD control samples (grey) (bottom left). Bottom right: box plots of summary MHC-I single-cell transcriptional score split by diagnosis. (D) Summary/average MHC-I score in individual IEC subtypes comparing CD, UC and controls. (E) Nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) expression in TI IEC of patients with CD colocalises with CD8+ T cells. RNA scope of TI biopsies from healthy donors and patients with CD. EPCAM (cyan), NLRC5 (white), TAP1 (yellow), CD8A (red), IFNG (green) and nuclei (DAPI, blue). Proximity of CD8 + T-cells with NLRC5 + EPCAM + cells in the CD biopsy is shown with arrows. Representative images are shown. Scale bar=100 µm and zoom in scale bar=10 µm.

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, Expressing, Control, Biomarker Discovery, Binding Assay, RNAscope

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Intestinal epithelial cells (IECs) present antigen via major histocompatibility complex class I (MHC-I) and activate CD8 + T cells in vitro with nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) acting as key modulator of mucosal inflammation in vivo. (A) Overview of experimental set-up. (B) Quantification of H2K b -SIINFEKL and pan-H2K b flow cytometry on live EpCAM + cells in murine intestinal epithelial organoids (IEOs) stimulated with or without interferon γ (IFNγ) (48 hours) and pulsed with or without OVA257–264 peptide (SIINFEKL) peptide. Data are representative of two independent experiments run in triplicates. GMFI, geometric mean fluorescence intensity; AU, arbitrary units. P values were calculated by two-way analysis of variance (ANOVA) with Bonferroni test for multiple comparisons (**p<0.01, ****p<0.0001). (C) Overview of experimental design. (D) Quantitative PCR gene expression of Ifng for coculture experiment in murine IEOs±SIINFEKL peptide pulse and cocultured with SIINFEKL-activated OTI T-cells. Data are presented as fold change over unstimulated OTI cells minus murine IEOs, normalised to Cd8a . P values were calculated using two-way ANOVA with Bonferroni’s multiple comparisons test (***p<0.001, ns=not significant). (E) Body weight changes over time during and after a 6-day course of 2% dextran sulphate sodium (DSS) exposure. (n=8 and n=5 Nlrc5fl/fl and Nlrc5-/- mice, respectively. P values calculated by multiple t-tests with Holm-Šídák correction for multiple comparisons.) (F) Quantification of H2K b surface expression on EpCAM+ cell populations within the lamina propria extractions of DSS-treated mice. All panels: data are representative of two independent experiments (**p<0.01). (G) Colon weight per unit length and mesenteric lymph node (MLN) weight and spleen weight of Nlrc5 wild type and knockout mice, on day 14 after initiation of 6-day course of 2% DSS (**p<0.01).

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, In Vitro, Binding Assay, In Vivo, Flow Cytometry, Fluorescence, Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Knock-Out

Journal: Cell reports

Article Title: Yap/Taz inhibit goblet cell fate to maintain lung epithelial homeostasis

doi: 10.1016/j.celrep.2021.109347

Figure Lengend Snippet: (A) Representative YAP/TAZ and MUC5AC immunostaining of human airway sections (performed using an antibody recognizing YAP and TAZ). DAPI-stained nuclei are in blue. Nuclei within MUC5AC + cells are highlighted with a yellow dotted line and with a blue dotted line in MUC5AC − cells. A white dotted line marks the basal surface of the epithelium (scale bars, 10 μm). (B) Quantification of nuclear YAP/TAZ intensity in airway epithelial cells across multiple sections from two patient donors. Cells were scored as either MUC5AC-positive or -negative and the intensity of YAP/TAZ staining was measured within the nuclear area outlined by DAPI staining (minimum of n = 14; unpaired t test, ****p < 0.0001). (C) YAP/MUC5AC immunostaining of human ALI cultures imaged by confocal microscopy. A z stack view is shown in the top panels (scale bars,10 μm). (D) HBECs were transfected with control siRNA (siCTL) or siRNA targeting YAP/TAZ (siY/T). MUC5AC, SCGB1A1, YAP , and WWTR1/TAZ qPCR analysis of lysates collected 72 h after knockdown (n = 6; unpaired t test, **p = 0.001, ****p < 0.0001). (E) Heatmap of gene expression changes resulting from YAP/TAZ knockdown in HBECs analyzed by RNA sequencing (RNA-seq). 2 distinct patient isolates were treated with three independent siCTLs or siRNA targeting YAP/TAZ (siY/T), and global gene expression changes were examined by RNA-seq after 48 h of culture (n = 3 per condition, 2-fold change cutoff, FDR = 0.05). (F) Pathway enrichment of significantly upregulated and downregulated genes following YAP/TAZ depletion in human airway epithelial cells identified by GSEA. Both the −log 10 p value and the percentage representation within each gene set are displayed. In all bar plots data are represented as mean ± SEM. See also and and .

Article Snippet: Mouse Wwtr1 Taqman probe Mm01289583_m1 , ThermoFisher , 4331182.

Techniques: Immunostaining, Staining, Confocal Microscopy, Transfection, Control, Knockdown, Gene Expression, RNA Sequencing

Journal: Cell reports

Article Title: Yap/Taz inhibit goblet cell fate to maintain lung epithelial homeostasis

doi: 10.1016/j.celrep.2021.109347

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse Wwtr1 Taqman probe Mm01289583_m1 , ThermoFisher , 4331182.

Techniques: Recombinant, Lysis, TUNEL Assay, RNAscope, Positive Control, Negative Control, Staining, Transfection, Purification, cDNA Synthesis, SYBR Green Assay, Knock-Out, Microarray, Knockdown, Control, Software

Journal: Cell Metabolism

Article Title: SARS-CoV-2 infects human pancreatic β cells and elicits β cell impairment

doi: 10.1016/j.cmet.2021.05.013

Figure Lengend Snippet: SARS-CoV-2-associated receptors are expressed in pancreatic β cells (A) Representative double immunofluorescence staining of ACE2, TMPRSS2, NRP1, and TFRC with the β cell marker, insulin (INS), and α cell marker, glucagon (GLU), in the normal human pancreas, donor 1. See . (B) Quantification of ACE2, TMPRSS2, NRP1, and TFRC in β cells (INS +) and α cells (GLU +) from a normal pancreas. No statistically significant changes in ACE2 and TMPRSS2 expression were detected between β and α cells. NRP1 and TFRC expression was statistically significantly higher in β cells compared with α cells. Rabbit anti-NRP1 (Abcam, ab81321, 1:200) and mouse anti-TFRC (Thermo Fisher, # 13-6800, 1:200) were used for the experiments shown here. Error bars represent mean ± SD (~10–15 islets from the pancreas of 5 non-COVID-19 donors; see ). ∗∗ p < 0.001, one-way ANOVA with Tukey’s post-test. Each dot represents one donor. Scale bars, 5 μm (A) and 2 μm (insets). See also and and .

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for SARS-CoV-2 spike mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Double Immunofluorescence Staining, Marker, Expressing

Journal: Cell Metabolism

Article Title: SARS-CoV-2 infects human pancreatic β cells and elicits β cell impairment

doi: 10.1016/j.cmet.2021.05.013

Figure Lengend Snippet: SARS-CoV-2 preferentially infects β cells of human pancreatic islets ex vivo (A–D) Mock-treated or SARS-CoV-2-infected human pancreatic islets were stained after 2 or 6 dpi. (A) Representative double immunofluorescence staining of SARS-CoV-2 nucleocapsid protein (NP) in combination with β cell marker, insulin (INS); ɑ cell marker, glucagon (GLU); δ cell marker, somatostatin (SST); and endothelial cell marker (CD31). (B) Representative double immunofluorescence staining of SARS-CoV-2 spike protein (SP) in combination with a similar combination of markers as (A). The nuclei were stained using DAPI (blue) as a counterstain. (C) Quantified percentages of SARS-CoV-2 NP and SP within α, β, δ, and endothelial cells of pancreatic islets. Around 40% to 60% NP and SP staining, respectively, are present within β cells. (D) Quantified percentages of SARS-CoV-2 NP- and SP-positive α, β, δ, and endothelial cells. (C and D) Error bars represent mean ± SD (~500–1,000 cells were quantified from healthy isolated human islets from donors 1–5; see ). (E) Representative double immunofluorescence staining of SARS-CoV-2 NP in combination with insulin after pre-treating islets with dimethyl sulfoxide (DMSO) or 100 μM EG00229 for 1 h before infection with SARS-CoV-2. Islets were fixed at 2 dpi and stained for SARS-CoV-2 NP and β cell marker, insulin (INS). Quantification of the percentages of β cells containing NP-positive β cells (right). Error bars represent mean ± SD (~500–1,000 cells were quantified from healthy isolated human islets from donors 10–13; see ). ∗ p < 0.05, two-tailed Student’s t test. Each dot represents one donor. Scale bars, 5 μm (A, B, and E) and 2 μm (insets). See also .

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for SARS-CoV-2 spike mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Ex Vivo, Infection, Staining, Double Immunofluorescence Staining, Marker, Isolation, Two Tailed Test

Journal: Cell Metabolism

Article Title: SARS-CoV-2 infects human pancreatic β cells and elicits β cell impairment

doi: 10.1016/j.cmet.2021.05.013

Figure Lengend Snippet: SARS-CoV-2 infects pancreatic β cells of patients with COVID-19 (A) Representative double immunofluorescence staining of pancreatic islets from patients with COVID-19 and healthy controls using antibodies against SARS-CoV-2 NP and INS. (B) Representative multiplexed images of in situ hybridization against the SARS-CoV-2 spike mRNA, in combination with immunofluorescence staining of insulin (INS). SARS-CoV-2 spike mRNA expression (red dots) was detected within pancreatic β cells. The nuclei were stained using DAPI (blue) as a counterstain. Scale bars, 5 μm (A and B) and 2 μm (insets). See also Figure S3 and .

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for SARS-CoV-2 spike mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Double Immunofluorescence Staining, In Situ Hybridization, Immunofluorescence, Staining, Expressing

Journal: Cell Metabolism

Article Title: SARS-CoV-2 infects human pancreatic β cells and elicits β cell impairment

doi: 10.1016/j.cmet.2021.05.013

Figure Lengend Snippet: SARS-CoV-2 infection interferes with insulin content/secretion and induces β cell apoptosis (A–F) Pancreatic islet functionality was analyzed by insulin content, glucose-stimulated insulin secretion (GSIS), and TUNEL staining ex vivo . (A) Insulin content is decreased in SARS-CoV-2-infected islets compared with mock-treated islets. (B) GSIS is decreased in SARS-CoV-2-infected islets compared with mock-treated islets. (A and B) Error bars represent mean ± SD (data were collected from 7 healthy isolated human islets, donors 2–8; see ). ∗ p < 0.05, two-tailed Student’s t test. (C) Representative staining of β cell apoptosis by in situ TUNEL and DAPI staining in β cells (INS) of mock- or SARS-CoV-2-treated human islets. DNase-treated sections were used as a positive control in the TUNEL assay. (D and F) Quantification of the percentages of islets containing TUNEL-positive β cells. Error bars represent mean ± SD (~500–1,000 cells were quantified from each of 3–5 separate healthy isolated human islets, donors 1–5 [D] and 7–9 [F]; see ). (E) Representative staining of β cell apoptosis by in situ TUNEL and DAPI staining in β cells (INS) of mock-treated versus SARS-CoV-2-SP-treated human islets. ∗ p < 0.05, ∗∗ p < 0.01, two-tailed Student’s t test. Scale bars, 5 μm (C and E). See also and , , , and .

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for SARS-CoV-2 spike mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Infection, TUNEL Assay, Staining, Ex Vivo, Isolation, Two Tailed Test, In Situ, Positive Control

Journal: Cell Metabolism

Article Title: SARS-CoV-2 infects human pancreatic β cells and elicits β cell impairment

doi: 10.1016/j.cmet.2021.05.013

Figure Lengend Snippet:

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for SARS-CoV-2 spike mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Virus, Recombinant, Blocking Assay, Enzyme-linked Immunosorbent Assay, In Situ, Control, RNAscope, Multiplex Assay, Purification, Expressing, Infection, Software