Journal: Diabetes

Article Title: Expression and Regulation of Chemokines in Murine and Human Type 1 Diabetes

doi: 10.2337/db11-0853

Figure Lengend Snippet: Chemokine transcripts induced in human islet cells in response to inflammatory stimuli (microarray analysis). Purified human islets obtained from healthy organ donors were cultured for 24 h in the absence (Control) or presence of individual recombinant human cytokines IL-1β, TNFα, or IFNγ or combinations thereof (MIX) prior to microarray analysis as described in . The normalized intensity (log scale) from data obtained on the HG U133 Plus 2.0 Affymetrix chip is shown. Each data point is the mean ± SE of three to four observations. Cytokine cocktail (MIX)-induced expression by a factor of >30 was observed for CCL5 , CCL8 , CCL22 , CX3CL1 , CXCL9 , and CXCL10 (asterisks indicate significant differences between control and cytokine-treated islets).

Article Snippet: Subcloning of human chemokine cDNA clones (Open Biosystems) into pIRES2-AcGFP1 vector (Clontech), transfection, and culture of human embryonic kidney 293 cells and staining with chemokine-specific antibodies for flow cytometric analysis were performed using previously published protocols ( ).

Techniques: Microarray, Purification, Cell Culture, Recombinant, Expressing

Journal: Diabetes

Article Title: Expression and Regulation of Chemokines in Murine and Human Type 1 Diabetes

doi: 10.2337/db11-0853

Figure Lengend Snippet: Chemokine transcripts induced in human islet cells in response to inflammatory stimuli (qRT-PCR analysis). Chemokine transcript expression in human islets cultured as described in the legend to and was measured by qRT-PCR using a 5′-nuclease assay and FAM dye–labeled TaqMan MGB probes with two PCR primers. Endogenous HPRT1 was used for normalization. Data (mean ± SE; four donors) was quantified using the 2 –ΔΔ C T method and expressed relative to an islet sample incubated in medium alone. For direct comparison, a value of 1.0 (dotted line) was assigned to TNFα-induced ( CCL5 , CXCL9/10 , and CX3CL1 ) or IL-1β–induced ( CCL22 ) chemokine transcripts. Asterisks indicate significant differences between control and cytokine-treated islets.

Article Snippet: Subcloning of human chemokine cDNA clones (Open Biosystems) into pIRES2-AcGFP1 vector (Clontech), transfection, and culture of human embryonic kidney 293 cells and staining with chemokine-specific antibodies for flow cytometric analysis were performed using previously published protocols ( ).

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Nuclease Assay, Labeling, Incubation

Journal: Diabetes

Article Title: Expression and Regulation of Chemokines in Murine and Human Type 1 Diabetes

doi: 10.2337/db11-0853

Figure Lengend Snippet: Immunofluorescent localization of chemokines in cultured human islets. Islets were cultured for 24 h in MIX, fixed with 4% paraformaldehyde (PFA), embedded in paraffin, sectioned, and stained by the immunofluorescent procedure. Insulin (Cy2), glucagon (AMCA), and chemokine (Cy3) immunofluorescent reactivity are shown. Please note that a certain loss of insulin-staining intensity typically occurs as a result of the nature of our islet isolation and culture procedure; however, the integrity of β-cell function was verified in vivo as detailed in . The figure is representative of two experiments performed with two different donor islets. INS/GCG, insulin/glucagon; MERGE, merged images. Scale bar: 15 μm. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: Subcloning of human chemokine cDNA clones (Open Biosystems) into pIRES2-AcGFP1 vector (Clontech), transfection, and culture of human embryonic kidney 293 cells and staining with chemokine-specific antibodies for flow cytometric analysis were performed using previously published protocols ( ).

Techniques: Cell Culture, Staining, Isolation, Cell Function Assay, In Vivo

Journal: Diabetes

Article Title: Expression and Regulation of Chemokines in Murine and Human Type 1 Diabetes

doi: 10.2337/db11-0853

Figure Lengend Snippet: Chemokine expression in the RIP-GP model of virus-induced type 1 diabetes. RIP-GP mice were infected with LCMV, and their pancreata were harvested 7 days later and processed for immunohistological analysis as detailed in . Note the minimal or absent expression of CCL22 and CXCL9, the preferential expression of CCL8 and CXCL10 by β-cells, as well as CX3CL1 production by α-cells; the right-hand column features magnified sections of merged CCL8, CXCL10, and CX3CL1 stains. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: Subcloning of human chemokine cDNA clones (Open Biosystems) into pIRES2-AcGFP1 vector (Clontech), transfection, and culture of human embryonic kidney 293 cells and staining with chemokine-specific antibodies for flow cytometric analysis were performed using previously published protocols ( ).

Techniques: Expressing, Infection

Journal: Diabetes

Article Title: Expression and Regulation of Chemokines in Murine and Human Type 1 Diabetes

doi: 10.2337/db11-0853

Figure Lengend Snippet: Chemokine expression in type 1 diabetic (T1D) and healthy control (Control) pancreata. Pancreatic sections from healthy control subjects (case identification nos. 6117, 6112, and 6115) and type 1 diabetic donors (case identification nos. 6052 and 6087) were acquired through the nPOD program and stained for insulin, glucagon, and chemokines as detailed in . Note the presence of some CCL5, CCL8, and CXCL9 in diabetic donors but their absence in healthy control samples. Only very faint CX3CL1 staining was observed in one of the type 1 diabetic samples (identification no. 6087). Scale bar: 20 μm. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: Subcloning of human chemokine cDNA clones (Open Biosystems) into pIRES2-AcGFP1 vector (Clontech), transfection, and culture of human embryonic kidney 293 cells and staining with chemokine-specific antibodies for flow cytometric analysis were performed using previously published protocols ( ).

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells

doi: 10.1038/s41598-017-17730-2

Figure Lengend Snippet: Microarray analysis shows that DF-1 have an attenuated innate response compared with CEFs. ( A ) Results from the microarray comparison between untreated DF-1 and CEFs. Pie charts represent the number of up- and down- regulated transcripts associated with different biological processes, assessed by Gene Ontology (GO) search and summarized according to their functions (PANTHER classification system). Scatter plots and Venn diagrams, showing extent of differential gene expression (Log2) and numbers of genes differentially regulated, respectively, in comparisons between CEFs and DF-1, either ( B ) treated with recombinant chIFN-α (1000 units/ml, 6 h), or ( C ) infected with IBDV (multiplicity of infection, MOI 5; 16 h). Scatter plots were generated using the Genespring scatter plot tool. Scatter plots show DF-1 (Y-axis) versus CEFs (X-axis) cells. The scale on the X- and Y-axis indicate expression levels (log2) and change in gene expression represented as a gradient of blue and red color for low- and high-expression intensity respectively. ( D ) Cluster analysis and heat-map showing differential expression of the top 45 ISGs (|fold change| ≥3.0 and FDR ≤0.01) as identified in CEFs following chIFN-α stimulation. ISGs were ranked by hierarchical clustering. Columns represent five comparisons, left to right: IBDV-infected CEFs and DF-1, chIFN-α-stimulated CEFs and DF-1 (each compared to their respective mock-treated control) and mock-treated DF-1 compared to mock-treated CEFs. Fold change in gene expression is represented by a blue (down-regulated) to red (up-regulated) colour intensity gradient. ( E ) Microarray expression data (log2 normalised intensity values ± Standard Deviation) for SOCS1 in, IBDV-infected, chIFN-α –treated or mock-treated CEFs or DF-1.

Article Snippet: Full-length chicken SOCS1 was also amplified from RNA using Q5 High-Fidelity DNA polymerase (New England Biolabs, USA) and primers containing Nco I and Eco RI and was cloned into pEF.pLink2 .

Techniques: Microarray, Comparison, Gene Expression, Recombinant, Infection, Generated, Expressing, Quantitative Proteomics, Control, Standard Deviation

Journal: Scientific Reports

Article Title: Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells

doi: 10.1038/s41598-017-17730-2

Figure Lengend Snippet: Relative suppression or induction of ISG transcription by overexpression or knockdown, respectively, of SOCS1. ( A – C ) DF-1 were transfected with control siRNA or siRNA specific for SOCS1 for 42 h and mock-treated or treated with chIFN-α (1000 units/ml) for 6 h. ( D – F ) DF-1 were transfected with either empty vector or an HA-tagged SOCS1 expression plasmid (SOCS1p) for 42 h and and mock-treated or treated with chIFN-α (1000 units/ml) for 6 h. Extracted total RNA was subjected to reverse transcription followed by quantitative PCR using specific primer sets for SOCS1 (A and D), Mx1 (B and E), IFIT5 (C and F) normalized against GAPDH (using the ΔΔCt method). Data in A-F are representative from three independent experiments. One-way ( A–F ) Anova with Bonferroni posthoc test were used to analyse the data. * P < 0.05, *** P < 0.001, **** P < 0.0001. ( G ) & ( H ) Immunoblots confirming expression of exogenous HA-tagged SOCS1 following transfection of DF-1 with either pcDNA4 (empty vector) or pcDNA4 expressing HA-tagged SOCS1 ( G ) and silencing of SOCS1 following transfection of DF-1 with the Flag-tagged SOCS1 construct and either siRNA for SOCS1 or a control siRNA ( H ). Panels (G) and (H) show cropped images of the immunoblots; full-length blots are presented in Supplementary Fig. .

Article Snippet: Full-length chicken SOCS1 was also amplified from RNA using Q5 High-Fidelity DNA polymerase (New England Biolabs, USA) and primers containing Nco I and Eco RI and was cloned into pEF.pLink2 .

Techniques: Over Expression, Knockdown, Transfection, Control, Plasmid Preparation, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Construct

Journal: Scientific Reports

Article Title: Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells

doi: 10.1038/s41598-017-17730-2

Figure Lengend Snippet: The SOCS box is not essential for ChSOCS1 inhibition of JAK/STAT signalling in DF-1. ( A ) Schematic representation of the architecture of SOCS1 protein, expression plasmids encoding wild-type (SOCS1 WT) and SOCS box deletion mutant (SOCS1 ΔBOX) and alignment of sequences (in boxes) of the KIR motif and the SOCS box domain of human, mouse and chicken SOCS1 proteins. ( B ) Luciferase reporter gene assay in DF-1 for chicken Mx1 and viperin promoters following transient transfection expression plasmids SOCS1 WT and SOCS1 ΔBOX, each with and without chIFN-α treatment. Two-way Anova with Bonferroni posthoc test were used to analyse the data. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( C ) Immunoblot confirming expression of Flag-tag in Flag-tagged expression plasmids encoding for wild-type (WT) SOCS1 and SOCS box deletion mutant (ΔBOX). No protein was observed when DF-1 were transfected with SOCS1 KIR deletion mutant (ΔKIR) plasmid. Panel (C) shows cropped images of the immunoblots; full-length blots are presented in Supplementary Fig. .

Article Snippet: Full-length chicken SOCS1 was also amplified from RNA using Q5 High-Fidelity DNA polymerase (New England Biolabs, USA) and primers containing Nco I and Eco RI and was cloned into pEF.pLink2 .

Techniques: Inhibition, Expressing, Mutagenesis, Luciferase, Reporter Gene Assay, Transfection, Western Blot, FLAG-tag, Plasmid Preparation

Journal: Scientific Reports

Article Title: Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells

doi: 10.1038/s41598-017-17730-2

Figure Lengend Snippet: Modulating levels of SOCS1 in CEFs and DF-1 regulates viral RNA expression and virus yield. ( A ) Titres of IBDV PBG98 recovered from CEFs and DF-1 after transient transfection with a SOCS1 expression plasmid or siSOCS1, respectively. Virus titres are the sum of cell-associated and extracellular virus, determined by plaque assay on CEFs (Mean ± SEM). ( B ) Fold change (percent) of IBDV VP4 RNA levels in CEFs and DF-1 after transient transfection with a SOCS1 expression plasmid or siSOCS1, respectively. Virus titres and viral RNA levels were compared with those from cells transfected with empty vectors or control siRNA, as appropriate. Data are representative from three independent experiments. An unpaired t-test with Welch’s correction (Two-tailed) was used to analyse the data. ** P < 0.01, **** P < 0.0001.

Article Snippet: Full-length chicken SOCS1 was also amplified from RNA using Q5 High-Fidelity DNA polymerase (New England Biolabs, USA) and primers containing Nco I and Eco RI and was cloned into pEF.pLink2 .

Techniques: RNA Expression, Virus, Transfection, Expressing, Plasmid Preparation, Plaque Assay, Control, Two Tailed Test

Journal: STAR Protocols

Article Title: Integration of SNP genotyping and 16S rRNA amplicon sequencing to identify heritable gut microbes in chickens

doi: 10.1016/j.xpro.2023.102071

Figure Lengend Snippet:

Article Snippet: QIAamp DNA Stool Mini Kit , Qiagen , Cat#51604.

Techniques: Amplification, Sequencing, Microarray, Software, Gas Phase Electrophoretic Molecular Mobility Analysis

Journal: Nature

Article Title: Loss of colonic fidelity enables multilineage plasticity and metastasis

doi: 10.1038/s41586-025-09125-5

Figure Lengend Snippet: (a) Targeted DNA sequencing of targeted Atrx locus in BPN Atrx KO organoid lines. (b) Representative pictures of BPN Control and BPN Atrx KO organoid culture. Scale bars, 1000 µm. (c) RT-qPCR analysis of the EMT marker Twist1 in BPN Control vs BPN Atrx KO organoid culture, n = 3 vs 3 independent experiments. (d) Fluorescence microscopy of phalloidin stained BPN Control and BPN Atrx KO organoids following treatment with 5 ng/ml TGF-beta. Zoomed area outlined in white box. Scale bars, 400 μm overview and 200 μm zoom. (e) Average number of clusters of BPN Control and BPN Atrx KO organoids per well exhibiting a spindle-like morphology following TGF-beta treatment, n = 5 vs 4 independent experiments. (f) RT-qPCR analysis of EMT and epithelial marker expression in BPN Atrx KO organoids either untreated or treated with 5 ng/ml TGF-beta, n = 3 vs 3 independent experiments. (g) Representative H&E and β-catenin-stained images of primary tumours in mice orthotopically transplanted with BPN Control or BPN Atrx KO organoid cells. β-catenin is used to identify tumour cells. Magnification highlights the distinct morphological features. Scale bars, 1 mm overview and 100 µm zoom, n = 6 vs 8 mice. (h) Quantification of tumour areas with glandular morphology in BPN Control and BPN Atrx KO primary tumours, n = 6 vs 8 mice. (i) Representative H&E-stained image of BPN Atrx KO metastasis. Scale bars, 1 mm. (j) Summary data indicating presence or absence of metastases. Number of mice with metastases or no metastases indicated on graph, n = 6 vs 8 mice. For (c) and (f) gene expression was normalised to Actb and relative levels were calculated using the ΔΔCt method. Data are mean ± SD. For (c), (e) and (f) p values were calculated using two-tailed student’s ttest. For (e) p = 0.000023. For (h) p value was calculated using two-tailed Mann-Whitney test. (k) Normalised enrichment scores of significantly enriched gene sets in TGF-beta treated AKP Control vs AKP Atrx KO organoids. (l) RT-qPCR analysis of representative EMT markers induced in AKP Atrx KO organoids following treatment with TGF-beta, n = 3 vs 3 independent experiments. (m) Normalised enrichment scores of significantly enriched gene sets in untreated AKP Control vs AKP Atrx KO organoids. (n) RT-qPCR analysis of representative genes important for colonic lineage specification and function, n = 3 vs 3 independent experiments. (o) RT-qPCR analysis of representative genes important for colonic lineage specification and function in BPN Control vs BPN Atrx KO organoids, n = 3 vs 3 independent experiments. For (l) gene expression was normalised to 18S rRNA and levels relative to untreated AKP control organoids calculated using the ΔΔCt method. For Twist1, Irx2 and Tfap2c gene expression was normalised to Actb and levels relative to untreated AKP control organoids calculated using the ΔΔCt method. For (n) gene expression was normalised to Actb and levels relative to untreated AKP Control organoids calculated using the ΔΔCt method. For (o) gene expression was normalised to Actb and levels relative to BPN Control organoids calculated using the ΔΔCt method. For (l) and (n) data are mean ± SD. P values were calculated using ordinary one-way ANOVA with multiple comparisons. For (l) Fgf2 p = 0.00000006, Serpine2 p = 0.000006, Col1a1 p = 0.000005, Col6a1 p = 0.000045, Col6a2 p = 0.000004, Pcolce p = 0.000015, Twist1 control p = 0.00000026, Twist1 + TGFβ1 p = 0.00000004. For (n) Hnf4a control p = 0.00000001, Cdx1 control p = 0.000000000000051, Cftr control p = 0.00000008. For (o) data are mean ± SD. P values were calculated using two-tailed student’s ttest. (p) Representative images of LY6D, KRT5 and EPCAM stained oesophagus and colon from Human Protein Atlas (Human Protein Atlas proteinatlas.org).

Article Snippet: The following primary antibodies were used: KRT5 (rabbit; Abcam 52635; 1:200 or chicken; BioLegend 905903; 1/200); LY6D (rabbit; Atlas HPA024755; 1:200); TWIST (mouse; Santa Cruz 81417, 1:200); EPCAM (rabbit; Abcam 71916; 1:200); HNF4A (rabbit; CST 3113; 1:500); ATRX (mouse; Sigma MABE1798; 1:500); β-catenin (mouse; BD 610154; 1:50); E-cadherin (rabbit; CST 3195, 1:200); and CDX2 (mouse; Atlas AMAb 91828, 1:1,000).

Techniques: DNA Sequencing, Control, Quantitative RT-PCR, Marker, Fluorescence, Microscopy, Staining, Expressing, Gene Expression, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Loss of colonic fidelity enables multilineage plasticity and metastasis

doi: 10.1038/s41586-025-09125-5

Figure Lengend Snippet: a , FACS plots for analysing and sorting EPCAM + LY6D + cells from AKP control and AKP Atrx KO organoids. b , Quantification of the percentage of cells in each EPCAM and LY6D population ( n = 3 independent experiments each). c , Quantification of the percentage of LY6D + cells in AKP control and AKP Atrx KO organoids ( n = 3 independent experiments each). P = 0.000019. d , RT–qPCR analysis of squamous cell markers in LY6D – and LY6D + cells sorted from AKP Atrx KO organoids ( n = 3 independent experiments each). Gene expression was normalized to Actb , and levels relative to LY6D – cells were calculated using the ΔΔ C t method. e , Representative images of KRT5-stained subcutaneous tumours and lung metastases from mice injected with AKP control or AKP Atrx KO organoids. f , Quantification of the percentage of KRT5 + cells in subcutaneous tumours from mice injected with AKP control or AKP Atrx KO organoids ( n = 5 mice each). g , Quantification of the percentage of KRT5 + cells in subcutaneous tumours and lung metastases from mice injected with AKP Atrx KO organoid cells ( n = 5 (subcutaneous) and 6 (lung metastasis) mice). h , Representative images of EPCAM and KRT5 co-immunofluorescence in AKP Atrx KO subcutaneous tumours. White arrows indicate cells exclusively expressing EPCAM (in EPCAM only panel, magenta), KRT5 (in KRT5 only panel, green) or co-expressing EPCAM and KRT5 (merge). n = 3 biologically independent samples. i , FACS plots for analysing and sorting LY6D + ITGA5 + cells from AKP control and AKP Atrx KO organoids. j , Quantification of the percentage of cells in each LY6D and ITGA5 population ( n = 3 independent experiments each). k , Schematic of the strategy used to determine the plasticity of different ITGA5 and LY6D expressing cell populations in AKP Atrx KO organoids. l , Quantification of the percentage of cells in each LY6D and ITGA5 population 9 days after plating. The original plated population is noted on the x axis ( n = 4 independent experiments each). m , Representative images of KRT5-stained subcutaneous tumours from mice injected with different LY6D and ITGA5 populations derived from AKP Atrx KO organoid cells. The population transplanted is indicated above each image ( n = 5 mice each). Scale bars, 50 µm. Data are the mean ± s.d. ( c , d , f , g ). P values were calculated using two-tailed Student’s t -test ( c , d ) or two-tailed Mann–Whitney test ( f , g ). Scale bars, 50 µm ( e , h , m ). The schematic in k was created using BioRender ( https://biorender.com ).

Article Snippet: The following primary antibodies were used: KRT5 (rabbit; Abcam 52635; 1:200 or chicken; BioLegend 905903; 1/200); LY6D (rabbit; Atlas HPA024755; 1:200); TWIST (mouse; Santa Cruz 81417, 1:200); EPCAM (rabbit; Abcam 71916; 1:200); HNF4A (rabbit; CST 3113; 1:500); ATRX (mouse; Sigma MABE1798; 1:500); β-catenin (mouse; BD 610154; 1:50); E-cadherin (rabbit; CST 3195, 1:200); and CDX2 (mouse; Atlas AMAb 91828, 1:1,000).

Techniques: Control, Quantitative RT-PCR, Gene Expression, Staining, Injection, Immunofluorescence, Expressing, Derivative Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Loss of colonic fidelity enables multilineage plasticity and metastasis

doi: 10.1038/s41586-025-09125-5

Figure Lengend Snippet: (a) RT-qPCR analysis of Emp1 and Lgr5 expression in LY6D +ve vs LY6D -ve cells sorted from AKP Atrx KO organoids, n = 3 vs 3 independent experiments. (b) RT-qPCR analysis of squamous markers Ly6d and Krt5 expression in tumours from mice injected with AKP Control or AKP Atrx KO organoids, n = 5 vs 5 tumours. (c) Representative images of KRT5 staining of AKP Atrx KO subcutaneous tumours (left) and lung metastases (right). Examples of the different morphologies of KRT5+ cells observed in these tumours are shown. In subcutaneous example 1, stratified epithelium (indicated by a black *) and elongated cells (indicated by black arrow) are shown. In subcutaneous tumour example 2 a keratin pearl is shown. In lung metastasis example 1 elongated cells are shown (indicated by black arrows). In lung metastasis example 2 stratified regions are shown. (d) Quantification of percentage of ITGA5 +ve single positive, LY6D +ve single positive, and ITGA5 +ve /LY6D +ve double positive cells in AKP Control vs AKP Atrx KO organoids, n = 3 vs 3 independent experiments. For (a) gene expression was normalised to Actb and levels relative to LY6D -ve cells calculated using the ΔΔCt method. Data are mean ± SD. P values were calculated using two-tailed student’s ttest. For (b) gene expression was normalised to Actb and levels relative to AKP Control tumours calculated using the ΔΔCt method. Data are mean ± SD. P values were calculated using two-tailed student’s ttest. For Ly6d p = 0.00005. For (d) data are mean ± SD. P values were calculated using two-tailed student’s ttest. For ITGA5+ cells (left panel) p = 0.000044. (e) Quantification of percentage of ITGA5 -ve /LY6D -ve double negative, LY6D +ve single positive, ITGA5 +ve single positive, and ITGA5 +ve / LY6D +ve double positive cells in cultures of AKP Atrx KO organoids 9 days after plating of different sorted populations. The plated population is indicated on the x-axis and the population being analysed indicated in the graph title and y-axis, n = 4 vs 4 independent experiments. (f) Quantification of percentage of KRT5+ cells in tumours derived from mice injected with ITGA5 -ve /LY6D -ve double negative, LY6D +ve single positive, ITGA5 +ve single positive, or ITGA5 +ve / LY6D +ve double positive AKP Atrx KO cells, n = 5 vs 5 mice. For (e) and (f) data are mean ± SD. P values were calculated using one-way ANOVA with multiple comparisons test.

Article Snippet: The following primary antibodies were used: KRT5 (rabbit; Abcam 52635; 1:200 or chicken; BioLegend 905903; 1/200); LY6D (rabbit; Atlas HPA024755; 1:200); TWIST (mouse; Santa Cruz 81417, 1:200); EPCAM (rabbit; Abcam 71916; 1:200); HNF4A (rabbit; CST 3113; 1:500); ATRX (mouse; Sigma MABE1798; 1:500); β-catenin (mouse; BD 610154; 1:50); E-cadherin (rabbit; CST 3195, 1:200); and CDX2 (mouse; Atlas AMAb 91828, 1:1,000).

Techniques: Quantitative RT-PCR, Expressing, Injection, Control, Staining, Gene Expression, Two Tailed Test, Derivative Assay

Journal: Nature

Article Title: Loss of colonic fidelity enables multilineage plasticity and metastasis

doi: 10.1038/s41586-025-09125-5

Figure Lengend Snippet: (a) Representative images of AKP Control and AKP Atrx KO cells treated or untreated with 5 ng/ml TGF-beta (TGFβ) for 72 h, n = 3 vs 3 independent experiments. Scale bars, 200 μm. (b) Relative cell viability of AKP Control vs AKP Atrx KO single cells treated or untreated with TGF-beta, n = 3 vs 3 independent experiments. (c) Targeted DNA sequencing of targeted Tgfbr2 locus in AKP Atrx KO organoid lines. (d) RT-qPCR analysis of representative genes important for squamous, EMT and colonic lineage specification and function in AKP Control, AKP Atrx KO Control and AKP Atrx KO Tgfbr2 KO organoids. (e) FACS plots for analysing LY6D +ve /ITGA5 +ve cells from AKP Atrx KO Control and AKP Atrx KO Tgfbr2 KO organoids, n = 3 vs 3 independent experiments. (f) Quantification of percentage of cells in each LY6D / ITGA5 population, n = 3 vs 3 independent experiments. (g) Fluorescence microscopy of phalloidin stained AKP Atrx KO Control and AKP Atrx KO Tgfbr2 KO organoids following treatment with 5 ng/ml TGF-beta, n = 3 vs 3 independent experiments. Scale bars, 200 μm. (h) Quantification of presence of spindle -like organoid structures formed following treatment of AKP Atrx KO Control and AKP Atrx KO Tgfbr2 KO organoids with 5 ng/ml TGF-beta, n = 3 vs 3 independent experiments. For (b) data are presented as mean values ± SD. P values were calculated using one-sided ANOVA multiple comparisons test. For (d) n = 1 biologically independent sample examined over 3 independent experiments. Gene expression was normalised to Actb and levels relative to AKP Control calculated using the ΔΔCt method. Data are mean ± SD. P values were calculated using one-sided ANOVA multiple comparisons test. For (h) p value was calculated using two-tailed student’s ttest.

Article Snippet: The following primary antibodies were used: KRT5 (rabbit; Abcam 52635; 1:200 or chicken; BioLegend 905903; 1/200); LY6D (rabbit; Atlas HPA024755; 1:200); TWIST (mouse; Santa Cruz 81417, 1:200); EPCAM (rabbit; Abcam 71916; 1:200); HNF4A (rabbit; CST 3113; 1:500); ATRX (mouse; Sigma MABE1798; 1:500); β-catenin (mouse; BD 610154; 1:50); E-cadherin (rabbit; CST 3195, 1:200); and CDX2 (mouse; Atlas AMAb 91828, 1:1,000).

Techniques: Control, DNA Sequencing, Quantitative RT-PCR, Fluorescence, Microscopy, Staining, Gene Expression, Two Tailed Test

Journal: Nature

Article Title: Loss of colonic fidelity enables multilineage plasticity and metastasis

doi: 10.1038/s41586-025-09125-5

Figure Lengend Snippet: a) PCA plot of ATAC-seq data comparing AKP Control vs AKP Atrx KO organoids, n = 3 vs 3 independent experiments. (b) Representative IGV browser tracks of ATAC peaks at colonic epithelial genes. Significantly altered peaks are outlined with a red box. Blue track refers to AKP control , while red track refers to AKP Atrx KO organoids. (c) Representative IGV browser tracks of ATAC peaks at mesenchymal genes. Significantly altered peaks are outlined with a red box. Blue track refers to AKP control , while red track refers to AKP Atrx KO organoids. (d) Representative IGV browser tracks of ATAC peaks at squamous genes. Significantly altered peaks are outlined with a red box. Note lack of significantly different peaks at Krt5 and Ly6d despite altered expression. Blue track refers to AKP control , while red track refers to AKP Atrx KO organoids. (e) Heatmap showing transcription factor binding sites enriched in regions that lose accessibility in AKP Atrx KO organoids. Brown shade gradient indicates regions where accessibility is lost in AKP Atrx KO organoids. (f) PCA plot of H3K27ac CUT&RUN comparing AKP Control vs AKP Atrx KO organoids, n = 3 vs 3 independent experiments. (g) RT-qPCR analysis of colonic epithelial cell marker expression in AKP Control , AKP Atrx KO and AKP Atrx KO Hnf4a overexpressing organoids, n = 3 vs 3 independent experiments. (h) RT-qPCR analysis of Hnf4α in AKP Control vs AKP Hnf4a KO organoids, n = 3 vs 3 independent experiments (i) Western-blot analysis of AKP Control and AKP Hnf4a KO organoids for HNF4A and β-actin. n = 4 vs 4 technical replicates. (j) Volcano plot depicting differentially expressed genes in AKP Control vs AKP Hnf4a KO organoids following RNAseq analysis. Red dots represent genes expressed at higher levels in the AKP Hnf4a KO organoids, while blue dots represent genes expressed at higher levels in the AKP Control , n = 3 vs 3 technical replicates. (k) Representative H&E images of subcutaneous tumours in mice subcutaneously injected with AKP Control or AKP Hnf4a KO organoid cells, n = 5 vs 5 mice. Magnification highlights the distinct morphological features. Scale bars, 1 mm overview and 250 µm zoom. (l) Quantification of non-glandular morphology area of AKP Control vs AKP Hnf4a KO subcutaneous tumours, n = 5 vs 5 mice. For (e) p values were calculated using one-sided Fisher’s exact test. For (g) and (h) gene expression was normalised to Actb and levels relative to AKP Control calculated using the ΔΔCt method. Data are mean ± SD. For (g) p values were calculated using one-way ANOVA with multiple comparisons test. For (h) p value was calculated using two-tailed student’s ttest. For (l) data are mean ± SD. P values were calculated using two-sided Mann-Whitney test.

Article Snippet: The following primary antibodies were used: KRT5 (rabbit; Abcam 52635; 1:200 or chicken; BioLegend 905903; 1/200); LY6D (rabbit; Atlas HPA024755; 1:200); TWIST (mouse; Santa Cruz 81417, 1:200); EPCAM (rabbit; Abcam 71916; 1:200); HNF4A (rabbit; CST 3113; 1:500); ATRX (mouse; Sigma MABE1798; 1:500); β-catenin (mouse; BD 610154; 1:50); E-cadherin (rabbit; CST 3195, 1:200); and CDX2 (mouse; Atlas AMAb 91828, 1:1,000).

Techniques: Control, Expressing, Binding Assay, Quantitative RT-PCR, Marker, Western Blot, Injection, Gene Expression, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Loss of colonic fidelity enables multilineage plasticity and metastasis

doi: 10.1038/s41586-025-09125-5

Figure Lengend Snippet: a , Representative IHC staining of a human CRC TMA for ATRX, HNF4A, CDX2 and LY6D. Examples of positive and negative staining are shown. Scale bar, 100 µm. b , Quantification of ATRX, HNF4A and CDX2 histoscore (H-score) values in LY6D – (<2% cells LY6D + ) and LY6D + (>2% cells LY6D + ) tumour cores. n = 500 (ATRX, HNF4A) and 509 (CDX2) biologically independent samples. c , Representative IHC image of a LY6D-stained human stage IV primary tumour. Scale bars, 50 µm. d , Summary data indicating the percentage of human primary tumours at stages I–III versus stage IV positive for LY6D. The percentages are based on >2% cells LY6D + and <2% cells LY6D – . P = 0.000027. e , Representative IHC image of LY6D-stained human liver metastasis. Scale bars, 100 µm. f , Summary data indicating the percentage of LY6D + cells in matched human primary tumours and liver metastases. n = 17 biologically independent matched samples (7 data points are visible as 11 primary tumour samples have the same value (0) and are overlapping). g , UMAP plot of Juanito scRNA-seq dataset overlayed with iCMS designation. For comparison, Atrx KO , Atrx WT and Atrx KO /Atrx WT transcriptional expression scores are overlayed in the same data. h , Survival plot of Marisa CRC patient dataset separated on Atrx -based gene expression clusters. i , Volcano plot of HOMER TF enrichment analysis of TFs with differential motif accessibilities between HiSquam and HiCol signature tumours. Selected TF motifs in regions with reduced accessibility in HiSquam tumours highlighted in blue and TF motifs in regions with increased accessibility in HiSquam tumours highlighted in red. Data are the mean ± s.d. ( b ). P values were calculated using two-tailed Mann–Whitney tests ( b ), two-sided Fisher’s exact tests ( d ), two-tailed Wilcoxon matched-pairs signed-rank tests ( f ), log-rank (Mantel–Cox) tests ( h ) or two-sided Fisher’s exact test and adjusted for multiple comparisons with the Benjamini–Yekutieli method ( i ). NS, not significant.

Article Snippet: The following primary antibodies were used: KRT5 (rabbit; Abcam 52635; 1:200 or chicken; BioLegend 905903; 1/200); LY6D (rabbit; Atlas HPA024755; 1:200); TWIST (mouse; Santa Cruz 81417, 1:200); EPCAM (rabbit; Abcam 71916; 1:200); HNF4A (rabbit; CST 3113; 1:500); ATRX (mouse; Sigma MABE1798; 1:500); β-catenin (mouse; BD 610154; 1:50); E-cadherin (rabbit; CST 3195, 1:200); and CDX2 (mouse; Atlas AMAb 91828, 1:1,000).

Techniques: Immunohistochemistry, Negative Staining, Staining, Comparison, Expressing, Gene Expression, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Loss of colonic fidelity enables multilineage plasticity and metastasis

doi: 10.1038/s41586-025-09125-5

Figure Lengend Snippet: (a) Scatter plot of individual tumour H-score values for HNF4A and ATRX of stained human CRC tissue microarray. (b) Scatter plot of individual tumour H-score values for CDX2 and ATRX of stained human CRC tissue microarray. (c) Scatter plot of individual tumour H-score values for CDX2 and HNF4A of stained human CRC tissue microarray. For (a), (b) and (c) samples staining positive for LY6D ( > 2% cells LY6D +ve ) highlighted in red. Data is individual value X/Y scatter plot. P values were calculated using two-sided Pearson’s correlation test. For (a) p < 0.000000000000001, (b) p = 0.0000000009, (c) p < 0.000000000000001. (d) Representative IHC staining of a human primary stage IV CRC tissue microarray for KRT5. (e) Summary data indicating the presence of KRT5 positive cells (>2% cells KRT5 +ve ) in human stage I-III vs stage IV primary CRC. (f) Representative IHC staining of a human matched liver metastasis tissue microarray for KRT5 expression. (g) Summary data indicating the percentage of KRT5 positive cells in matched human primary tumours and liver metastases. (h) Representative image of HNF4A IHC staining in matched human stage IV primary tumour and matched liver metastasis. Scale bars, 100 µm. (i) Summary data indicating the percentage of HNF4A positive cells in matched human primary tumours and liver metastases. (j) Representative image of ATRX IHC staining in matched human stage IV primary tumour and liver metastasis. Scale bars, 100 µm. (k) Summary data indicating the percentage of ATRX positive cells in matched human primary tumours and liver metastasis. For (e) p value was calculated using two-sided Fisher’s exact test. For (g), (i) and (k) p values were calculated using two-tailed Wilcoxon matched-pairs signed rank test. n = 17 vs 17 matched human primary stage IV tumours and liver metastases. (l) Targeted DNA sequencing of targeted ATRX locus in human CRC organoid line. (m) RT-qPCR analysis of colonic epithelial and squamous marker gene expression in human ATRX KO CRC organoids and control (NT), n = 3 vs 3 independent experiments. (n) Fluorescence microscopy of phalloidin stained human control ( NT ) and ATRX KO CRC organoids following treatment with 5 ng/ml TGF-beta, n = 3 vs 3 independent experiments. Zoomed area outlined in white box. Scale bars, 400 μm overview, 200 μm zoom. (o) Quantification of the percentage of human control ( NT ) and ATRX KO CRC organoids adopting spindle-like morphology following TGF-beta treatment. For (m) gene expression was normalised to ACTB . For (m) and (o) n = 1 biologically independent sample examined over 3 independent experiments. Data are mean ± SD. P values were calculated using two-tailed student’s ttest.

Article Snippet: The following primary antibodies were used: KRT5 (rabbit; Abcam 52635; 1:200 or chicken; BioLegend 905903; 1/200); LY6D (rabbit; Atlas HPA024755; 1:200); TWIST (mouse; Santa Cruz 81417, 1:200); EPCAM (rabbit; Abcam 71916; 1:200); HNF4A (rabbit; CST 3113; 1:500); ATRX (mouse; Sigma MABE1798; 1:500); β-catenin (mouse; BD 610154; 1:50); E-cadherin (rabbit; CST 3195, 1:200); and CDX2 (mouse; Atlas AMAb 91828, 1:1,000).

Techniques: Staining, Microarray, Immunohistochemistry, Expressing, Two Tailed Test, DNA Sequencing, Quantitative RT-PCR, Marker, Gene Expression, Control, Fluorescence, Microscopy

Journal: Nature

Article Title: Loss of colonic fidelity enables multilineage plasticity and metastasis

doi: 10.1038/s41586-025-09125-5

Figure Lengend Snippet: (a) Summary data indicating presence or absence of ATRX mutation in iCMS2 vs iCMS3 transcriptional subtypes. Data extracted from TCGA dataset. Number of tumours in each group indicated on graph, n = 348 vs 244 tumours. (b) Heatmap clustering of Marisa CRC patient dataset of 557 tumours based on squamous-like ( Atrx KO ) and colonic epithelial ( Atrx WT ) gene expression signatures. (c) Percentage of tumours located in the distal (left sided) or proximal (right sided) colon in different Atrx expression clusters. One-tailed Chi-squared test, p value = 1.72e-16. (d) MMR status of tumours in HiCol, Intermediate and HiSquam expression clusters. (e) Mutational status of tumours in HiCol, Intermediate and HiSquam expression clusters. (f) TNM stage of tumours in HiCol, Intermediate and HiSquam expression clusters. (g) Correlation of Atrx WT (colonic epithelial-like) and Atrx KO (squamous-like) gene expression signatures in GSE39582 dataset. iCMS status overlayed in purple (iCMS2) and orange (iCMS3). (h) Percentage of tumours designated as iCMS2 (i2) or iCMS3 (i3) transcriptional subtype in different Atrx expression clusters. Chi-squared test, p value = 2.2e-16. (i) FACS plots for analysing EPCAM +ve /LY6D +ve cells from normal colon and colon cancer samples. Box highlights the presence of EPCAM +ve /LY6D +ve cells. (j) Quantification of EPCAM +ve /LY6D +ve cells from normal colon and colon cancer samples, separated into left and right sided groups. For (a) p value was calculated using two-sided Fisher’s exact test. For (c), (d), (e), (f) and (h) p values were calculated using one-tailed Chi-Square test. For (g) Pearson’s correlation coefficient and two-sided p-value are described. The blue line and gray shaded area represent the regression line and 95% confidence interval. For (j) n = 5 (normal colon), n = 2 (left sided tumour), n = 3 (right sided tumour) biological independent samples. Data are mean ± SD. P values were calculated using ordinary one-way ANOVA with multiple comparisons.

Article Snippet: The following primary antibodies were used: KRT5 (rabbit; Abcam 52635; 1:200 or chicken; BioLegend 905903; 1/200); LY6D (rabbit; Atlas HPA024755; 1:200); TWIST (mouse; Santa Cruz 81417, 1:200); EPCAM (rabbit; Abcam 71916; 1:200); HNF4A (rabbit; CST 3113; 1:500); ATRX (mouse; Sigma MABE1798; 1:500); β-catenin (mouse; BD 610154; 1:50); E-cadherin (rabbit; CST 3195, 1:200); and CDX2 (mouse; Atlas AMAb 91828, 1:1,000).

Techniques: Mutagenesis, Gene Expression, Expressing, One-tailed Test

Journal: PLoS ONE

Article Title: Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients

doi: 10.1371/journal.pone.0127414

Figure Lengend Snippet: A: Western blot showing levels of dyskerin, NHP2 and NAF1 proteins in the iPS cells. All mutant cells show slightly decreased level of dyskerin protein compared to WT cells. Note that A353V iPS cells show decreased level of NHP2 while ΔL37 and Q31E cells don’t. The quantitive data, derived by densitometry, are shown. B: Real-time RT/PCR of RNA from iPS cells from DC patients and healthy controls. ΔL37 and Q31E mutant DKC1 iPS cells express same amount of DKC1 mRNA as WT iPS cells, but A353V mutant iPS cells show a slightly decreased level of DKC1 mRNA. GAPDH was used at loading control. The combined results of 4 independent experiments are shown, the error bars show standard deviation. C: Real-Time RT/PCR of TERC RNA (left) and TERT mRNA (right) in iPS cells. GAPDH was used at loading control. The combined results of 3 independent experiments are shown, the error bars show standard deviation. * p<0.01 between WT cells and different mutant cells, respectively. D: Telomerase activity assay of ΔL37 (left) and A353V (right) iPS cells compared to WT cells by using TRAP assay. 2×10 6 cells were extracted by using CHAPS lysis buffer and serial dilutions (50ng, 10ng, 2ng and 0.4ng) of each sample were assayed. IC: internal control, HI: heat inactivation. The quantitive data, derived by densitometry, are shown. E: Telomere length measurement of the iPS cells in different passages compared to those from the original fibroblast cells (Fib) by using pulse field gel electrophoresis and in-gel hybridization with telomere probe (TTAGGG) 3 .

Article Snippet: The donor construct was targeted to the AAVS1 locus using the AAVS1-SA-2A-puro-pA plasmid (Addgene) containing human DKC1 cDNA driven by the chicken β actin promoter.

Techniques: Western Blot, Mutagenesis, Derivative Assay, Quantitative RT-PCR, Control, Standard Deviation, Telomerase Activity Assay, TRAP Assay, Lysis, Nucleic Acid Electrophoresis, Hybridization

Journal: PLoS ONE

Article Title: Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients

doi: 10.1371/journal.pone.0127414

Figure Lengend Snippet: A: measurement of pseudouridine in 28S rRNA from WT and mutant iPS cells. iPS cells were labeled with 32 P-labeled orthophosphate and 28S rRNA was gel-purified. After digestion with RNase T 2 , each sample was separated by two-dimensional TLC. The positions of the labeled ribonucleotides are indicated. Ap: Adenine, Cp: Cytosine, Gp: Guanine, Up: Uridine, Ψp: Pseudouridine: B: Pulse–chase labeling experiments of rRNA isolated from WT , A353V and ΔL37 iPS cells. Cells were labeled with L-[ 3 H-methyl] methionine for 30 min and then chased in nonradioactive medium for the times shown. The RNA was separated on a 1.25% agarose gel, transferred to a nylon filter, and exposed to x-ray film. C: Real-time RT/PCR results of some H/ACA snoRNAs of WT and DKC1 mutant iPS cells. Results were expressed relative to GAPDH RNA. The combined results of 3 independent experiments are shown, the error bars show standard deviation.

Article Snippet: The donor construct was targeted to the AAVS1 locus using the AAVS1-SA-2A-puro-pA plasmid (Addgene) containing human DKC1 cDNA driven by the chicken β actin promoter.

Techniques: Mutagenesis, Labeling, Purification, Pulse Chase, Isolation, Agarose Gel Electrophoresis, Quantitative RT-PCR, Standard Deviation

Journal: PLoS ONE

Article Title: Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients

doi: 10.1371/journal.pone.0127414

Figure Lengend Snippet: A: Schematic representation of zinc-finger nuclease (ZNF)-mediated homologous recombination. The constitutively active AAVS1 “safe harbor” locus is shown on the top line and the targeting construct is shown below. The cDNA expression cassettes driving expression of Flag-tagged WT dyskerin under the chicken actin promoter (CAGG) were inserted by zinc finger-mediated homologous recombination into intron 1 of AAVS1 . HA, homologous arms left (L) and right (R); SA-2APuro-PA, puromycin drug resistance cassette. B: Western blot showing expression of flag-tagged dyskerin protein in corrected A353V and ΔL37 iPS cells by using anti-Flag antibody and anti-dyskerin antibody. Open arrow: endogenous dyskerin protein; filled arrow: Flag-tagged WT dyskerin protein. C: Northern blot result of TERC RNA expression levels of DKC1 corrected iPS cells. Different corrected lines are shown as well as the uncorrected line carrying the DKC1 ΔL37 or the DKC1 A353V mutation. β-actin was used as loading control. The quantitive data, derived by densitometry, are shown. D: The TRAP assay was performed to measure the telomerase activity after expressing the WT DKC1 gene in A353V and ΔL37 iPS cells, uncorrected and corrected by ectopic expression of WT DKC1 (DKC1-C) . The quantitive data, derived by densitometry, are shown E: Telomere lengths of DKC1 corrected iPS cells were measured by using in-gel hybridization with a telomere probe (TTAGGG) 3 . DKC1-C indicates corrected iPS cells expressing WT DKC1 gene.

Article Snippet: The donor construct was targeted to the AAVS1 locus using the AAVS1-SA-2A-puro-pA plasmid (Addgene) containing human DKC1 cDNA driven by the chicken β actin promoter.

Techniques: Zinc-Fingers, Homologous Recombination, Construct, Expressing, Western Blot, Northern Blot, RNA Expression, Mutagenesis, Control, Derivative Assay, TRAP Assay, Activity Assay, Hybridization

Journal: PLoS ONE

Article Title: Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients

doi: 10.1371/journal.pone.0127414

Figure Lengend Snippet: Fold changes of WNT related genes in DKC1 mutant iPS cells.

Article Snippet: The donor construct was targeted to the AAVS1 locus using the AAVS1-SA-2A-puro-pA plasmid (Addgene) containing human DKC1 cDNA driven by the chicken β actin promoter.

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients

doi: 10.1371/journal.pone.0127414

Figure Lengend Snippet: A: Validation of the results from microarray. LGR5 , DKK1 , WLS and FRZB mRNA expression were measured by real-time RT/PCR. 3 independent experiments were performed and the error bars show standard deviation. TERT CP: iPS cells with TERT R537H/2173-2187del15insACAG compound homozygotes mutation * p<0.01 between WT iPS cells and different mutant iPS cells, respectively. B: Western blot result of dyskerin protein in HEK293T cells showing doxycycline inducible DKC1 shRNA can reduce the dyskerin level to about 10–20%. shGFP was used as a control. C: Telomerase activity of HEK293T cells after knocking down of dyskerin protein from B was measured by TRAP assay. The quantitive data, derived by densitometry, are shown. D; Knock down of dyskerin protein can directly decrease the expression of LGR5 , WLS and FRZB genes. Two different DKC1 shRNA lines were used in this experiment. The combined results of 3 independent experiments are shown, the error bars show standard deviation. * p<0.01.

Article Snippet: The donor construct was targeted to the AAVS1 locus using the AAVS1-SA-2A-puro-pA plasmid (Addgene) containing human DKC1 cDNA driven by the chicken β actin promoter.

Techniques: Biomarker Discovery, Microarray, Expressing, Quantitative RT-PCR, Standard Deviation, Mutagenesis, Western Blot, shRNA, Control, Activity Assay, TRAP Assay, Derivative Assay, Knockdown

Journal: PLoS ONE

Article Title: Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients

doi: 10.1371/journal.pone.0127414

Figure Lengend Snippet: A: Real-Time RT/PCR experiments showed that, in A353V iPS cells, the mRNA expression of LGR5 , FRZB and WLS was significantly increased after expressing WT dyskerin protein. The combined results of 3 independent experiments are shown, the error bars show standard deviation. * p<0.01. B: Knock down of dyskerin affects canonical WNT signaling. The relative luciferase activity in inducible DKC1 -shRNA HEK293T cells transfected with plasmids containing a luciferase gene under the control of a WT (OT-flash) or mutant (OF-flash) β-catenin responsive promotor and pRL-SV40 plasmid. Luciferase activity was measured by using dual-luciferase system according to protocol from manufacturer. OT: WT TCF/LEF reporter, OF: Mutant TCF/LEF reporter, Dox: Doxycycline, CHIR: CHIR-99021.Four independent experiments were carried out and the mean of results are shown; the error bars showing standard deviation. * p<0.01.

Article Snippet: The donor construct was targeted to the AAVS1 locus using the AAVS1-SA-2A-puro-pA plasmid (Addgene) containing human DKC1 cDNA driven by the chicken β actin promoter.

Techniques: Quantitative RT-PCR, Expressing, Standard Deviation, Knockdown, Luciferase, Activity Assay, shRNA, Transfection, Control, Mutagenesis, Plasmid Preparation

Journal: Genomics Data

Article Title: DNA microarray global gene expression analysis of influenza virus-infected chicken and duck cells

doi: 10.1016/j.gdata.2015.03.004

Figure Lengend Snippet: Genomic DNA (gDNA) based probe selection to improve the sensitivity of chicken Genechip for duck transcriptome analysis. (A) Anas platyrhynchos genomic DNA (gDNA) hybridization intensity thresholds used to generate the probe mask files is shown. Data were obtained by hybridizing duck gDNA on chicken Genechip. Number of Gallus gallus probe pairs and probe sets from the chicken GeneChip® array retained across a range of gDNA intensity threshold is shown. Probe pairs retained (data in blue) is scaled to the left hand y- axis, while number of probe sets retained (data in red) are scaled to the to the right-hand y- axis). Intensity threshold of 200 gave highest number of genes differentially regulated following 24 h of infection with influenza viruses (H2N3, 50–92 and ty-Ty) compared to mock-infected controls. (B) All the genes significantly differentially regulated ( p < 0.05). (C) Genes regulated ± 2-fold following infection. (D) Genes significantly regulated ± 2-fold ( p < 0.05).

Article Snippet: Briefly, Pekin duck ( A. platyrhynchos ) genomic DNA from cells was biotin-labelled and hybridized to the Chicken ( G. gallus ) GeneChip® array and a probe intensity data file ( cel ) was generated as described above.

Techniques: Selection, Hybridization, Infection

Journal: Genomics Data

Article Title: DNA microarray global gene expression analysis of influenza virus-infected chicken and duck cells

doi: 10.1016/j.gdata.2015.03.004

Figure Lengend Snippet:

Article Snippet: Briefly, Pekin duck ( A. platyrhynchos ) genomic DNA from cells was biotin-labelled and hybridized to the Chicken ( G. gallus ) GeneChip® array and a probe intensity data file ( cel ) was generated as described above.

Techniques: Transformation Assay, Virus, Microarray, Gene Expression, Infection

Journal: PLoS ONE

Article Title: Large Sex Differences in Chicken Behavior and Brain Gene Expression Coincide with Few Differences in Promoter DNA-Methylation

doi: 10.1371/journal.pone.0096376

Figure Lengend Snippet: Heat map columns represent individual microarrays, while rows represent significantly differentially expressed (DE) probesets adjusted for false discovery rate. Heat maps are organized using hierarchical cluster analysis (average linkage) identifying three major clades both within and across breeds (clades are represented by brackets). A ) Probesets DE within the Red Junglefowl (RJF pools). B ) Probesets DE within the White Leghorn (WL pools). C ) Venn-diagram showing numbers of DE probesets within breeds that are also DE in the other breed. D ) Probesets DE across breeds. *Note that DE probesets annotated to autosomes are common within breed, but only three probesets remain in the across breed comparison, all annotated to the same gene: ZFR on chromosome 1. Each microarray was hybridized with a pool of six birds.

Article Snippet: To evaluate the generality of our findings, we used two genetically distinct breeds with unique selection histories over several thousand years; the Red Junglefowl ( Gallus gallus ), ancestor of domestic chickens, and the domesticated White Leghorn ( Gallus gallus domesticus ).

Techniques: Comparison, Microarray

Journal: PLoS ONE

Article Title: Large Sex Differences in Chicken Behavior and Brain Gene Expression Coincide with Few Differences in Promoter DNA-Methylation

doi: 10.1371/journal.pone.0096376

Figure Lengend Snippet: Dots represent fold change differences (log 2) within three categories: A) and D) = Expression microarray probesets; B) and E) = DNA-methylation tiling array probes averaged within promoter; C) and F) = Individual DNA-methylation tiling array probes. A) shows gene expression differences on the Z chromosome. B) shows promoter methylation differences on the Z-chromosome. C) shows individual probe differences in the promoters of a novel gene close to MHM and CDC37L1 . D) shows gene expression differences on chromosome 1. E) shows promoter methylation differences on chromosome 1. F) shows individual probe differences in the promoter to ZFR on chromosome 1. Red dots signify down-regulation in females, and blue dots up-regulation. Clear dots indicate Red Junglefowl (RJF) and dark colored dots White Leghorn (WL). The clear green shaded area represents the dosage compensated region reported by , and the dark green line represent the location of the male hyper methylated region reported by . Genebuild: WASHUC2.

Article Snippet: To evaluate the generality of our findings, we used two genetically distinct breeds with unique selection histories over several thousand years; the Red Junglefowl ( Gallus gallus ), ancestor of domestic chickens, and the domesticated White Leghorn ( Gallus gallus domesticus ).

Techniques: Expressing, Microarray, DNA Methylation Assay, Gene Expression, Methylation

Journal: PLoS ONE

Article Title: Large Sex Differences in Chicken Behavior and Brain Gene Expression Coincide with Few Differences in Promoter DNA-Methylation

doi: 10.1371/journal.pone.0096376

Figure Lengend Snippet: Heat map columns represent individual tiling arrays, while rows represent significantly differentially methylated (DM) probes adjusted for false discovery rate. Heat maps are organized using hierarchical cluster analysis (average linkage) identifying two major clades both within and across breeds (represented by brackets). A ) Probes DM within Red Junglefowl (RJF pools). B ) Probes DM within White Leghorn (WL pools). C ) Venn-diagram showing how many of the DM probes within breeds that are present across, in both, breeds. D ) Probes DM across breeds. Lower case letters next to heat map rows indicate that multiple probes are significantly DM in the same promoter; the same letter indicates the same promoter. Note that “d” and “a” represent probes in the promoters of ZFR and the novel gene in MHM respectively. Each tiling array was hybridized with a pool of six birds.

Article Snippet: To evaluate the generality of our findings, we used two genetically distinct breeds with unique selection histories over several thousand years; the Red Junglefowl ( Gallus gallus ), ancestor of domestic chickens, and the domesticated White Leghorn ( Gallus gallus domesticus ).

Techniques: Methylation

Journal: PLoS ONE

Article Title: Large Sex Differences in Chicken Behavior and Brain Gene Expression Coincide with Few Differences in Promoter DNA-Methylation

doi: 10.1371/journal.pone.0096376

Figure Lengend Snippet: Top candidate genes related to sexually dimorphic behavior in chickens (Red Junglefowl).

Article Snippet: To evaluate the generality of our findings, we used two genetically distinct breeds with unique selection histories over several thousand years; the Red Junglefowl ( Gallus gallus ), ancestor of domestic chickens, and the domesticated White Leghorn ( Gallus gallus domesticus ).

Techniques: Methylation