Journal: Cell Reports

Article Title: Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

doi: 10.1016/j.celrep.2021.109101

Figure Lengend Snippet:

Article Snippet: For microarray analysis, RNA was extracted from THP1 or stimulated human CD4+ T cells using the RNeasy Mini kit (QIAGEN).

Techniques: Recombinant, Multiplex sample analysis, Cell Isolation, Activation Assay, Staining, Flow Cytometry, Expressing, Reverse Transcription, Transfection, TA Cloning, Plasmid Preparation, Methylation, Immunoprecipitation, Purification, DNA Library Preparation, Library Quantification, Control, Sequencing, Methylation Sequencing, Amplification, Software

Journal: PLoS ONE

Article Title: Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease

doi: 10.1371/journal.pone.0194036

Figure Lengend Snippet: If allele-specific DNA methylation (ASM) around heterozygous SNP A/C exists (which is hypermethylated around A allele and hypomethylated around C allele), SNP is called A/C in micro array before digestion and A/A after digestion by MSREs. Thus, the probes heterozygous in uncut genomic DNA and homozygous in MSREs-digested DNA indicate ASM around SNP. Because we expect methylation skew between two alleles, all heterozygous SNPs which the ratio of signal intensities given two alleles changed after digestion should be extracted. SNP, single-nucleotide polymorphisms; MSREs, methylation-sensitive restriction enzymes, which contain HpaII ( 5′-CˆCGG-3′ ), HhaI ( 5′-GCGˆC-3′ ), and AciI ( 5′-CˆCGC-3′ ).

Article Snippet: DNA from patients was digested using a cocktail of three MSREs: HpaII (5′-CˆCGG-3′), HhaI (5′-GCGˆC-3′), and AciI (5′-CˆCGC-3′) (New England BioLabs, Ipswich, USA), which in combination interrogate the methylation status of ~32.4% of CpG sites in the human genome [ ].

Techniques: DNA Methylation Assay, Microarray, Methylation

Journal: PLoS ONE

Article Title: Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease

doi: 10.1371/journal.pone.0194036

Figure Lengend Snippet: SNP, single-nucleotide polymorphism; MSRE, methylation-sensitive restriction enzyme; RAS, relative allele score; IBD, inflammatory bowel disease; (G), genomic DNA; (D), DNA digested MSREs; (U), whole-genome-amplified fully unmethylated DNA digested MSREs.

Article Snippet: DNA from patients was digested using a cocktail of three MSREs: HpaII (5′-CˆCGG-3′), HhaI (5′-GCGˆC-3′), and AciI (5′-CˆCGC-3′) (New England BioLabs, Ipswich, USA), which in combination interrogate the methylation status of ~32.4% of CpG sites in the human genome [ ].

Techniques: Methylation, Amplification

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: The molecular structure of SOX11. The SOX11 protein is composed of 441 amino acids. SOX11 contains two functional domains, the N-terminal HMG domain and the conserved TAD. SOX11, sex-determining region Y-related high-mobility-group box transcription factor 11; HMG, high mobility group; TAD, C-terminal transactivation domain.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Functional Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: Overview of SOX11 function reported in diverse types of cancer.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Expressing, Over Expression, Biomarker Discovery, Migration, Methylation, DNA Methylation Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: Overall carcinogenic actions of SOX11 on the hallmarks of tumor biology. SOX11 exerts tumor-stimulative effects through increasing cell proliferation, repressing cell differentiation, inducing angiogenesis, and promoting metastasis. BCL6, B-cell lymphoma 6; BNIP3, B-cell lymphoma 2 (BCL2)/adenovirus E1B 19 kDa protein-interacting protein 3; CIC, cancer-initiating cell; PAX5, Paired box protein 5; PDGFA, platelet-derived growth factor A; SETMAR, SET domain and mariner transposase fusion gene; SOX11, Sex-determining region Y-related high-mobility-group box transcription factor 11; TANK, TRAF family member-associated NF-κB activator.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Cell Differentiation, Derivative Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: Different prognostic significance of SOX11 in tumor cases.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Expressing, Microarray, Over Expression

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) Immunohistochemistry showing reduced methylated H3K79 levels (H3K79me2) that reflect loss of DOT1L activity in damaged areas from osteoarthritic patients (OA) as compared to their corresponding preserved areas and to cartilage from non-OA patients. Images are representative of images from four different patients. Scale bar, 400 μm. ( b ) Heat maps of differential mRNA expression determined by quantitative PCR in chondrocytes treated with DOT1L inhibitor EPZ-5676 (EPZ) or vehicle (V) from passage 0 (P0) until P2, and from preserved versus damaged areas in OA cartilage. The colour code represents the mean expression level of six and four independent patient samples respectively. ( c ) Immunoblot analysis showing decreased methylated H3K79 levels in mouse articular chondrocytes after intra-articular injection of EPZ into C57Bl/6 wild-type mouse knees. The image is representative of one experiment with protein extracts pooled from two or three mice per condition. Unprocessed original scans of blots are shown in . ( d , e ) C57/Bl6 wild-type mouse knees were injected with EPZ (5 mg kg –1 ) or vehicle and killed after 2 or 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( d ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( e ). One experiment was performed with n =10 and 5. Representative images from the 4 week evaluation are shown. * P <0.05 (two-tailed t -test). Error bars indicate mean±s.e.m.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Immunohistochemistry, Methylation, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) KEGG pathway enrichment analysis of microarray data obtained from human articular chondrocytes treated with EPZ-5676 or vehicle. Nominal P values by EASE modified Fisher Exact test using the DAVID analysis tool (see Methods section) are shown. n =5 independent patient-derived cell cultures. ( b ) Co-immunoprecipitation (Co-IP) using an anti-DOT1L antibody showing interaction between DOT1L and β -catenin in human articular chondrocytes, that is increased upon Wnt activation by LiCl and disrupted upon DOT1L inhibition. The image is representative of three experiments. ( c ) TOP/FOP reporter assay in human articular chondrocytes after Wnt stimulation by LiCl and DOT1L inhibition by EPZ. Activity is compared to untreated cells (dotted line). n =3 biologically independent experiments. *** P <0.001 by one-way ANOVA. ( d , e ) LEF1 , TCF1 and c-MYC expression measured by quantitative PCR in chondrocytes treated with EPZ-5676 and LiCl ( d ) or in LiCl-treated chondrocytes transfected with siRNA directed against DOT1L or scrambled siRNA (siDOT1L or siSCR, respectively) ( e ). Data are from one experiment with three technical replicates. ( f ) Immunohistochemistry demonstrating increased TCF1 levels in the articular cartilage of C57/Bl6 wild-type mice after injection of EPZ-5676. The images are representative of three different animals. Scale bar, 200 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Microarray, Modification, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Activation Assay, Inhibition, Reporter Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunohistochemistry, Injection

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: All experiments were performed in healthy human articular chondrocytes: treated as indicated with DOT1L inhibitor EPZ-5676, Wnt activator LiCl, SIRT1 antagonist EX527 or SIRT1 agonist SRT1720; or transfected with DOT1L or scrambled siRNA. All data are presented as mean±s.e.m. ( a ) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis of DOT1L and methylated H3K79 and ( b ) acetylated H3K9 (H3K9Ac) and methylated H3K4 (H3K4me3) as markers of active transcription on the transcriptional start site (TSS) of Wnt target genes. Data are from two to five experiments. ( c ) Expression levels of TCF1 Wnt target gene measured by quantitative PCR in chondrocytes transfected with indicated specific or scrambled siRNA (siSCR). Data are from one experiment with technical triplicates. ( d ) TCF1 expression measured by quantitative PCR in the presence of SIRT1 agonist and antagonist. Data from two experiments each with technical triplicates. ( e ) Co-IP analysis using the indicated antibodies demonstrating the interaction of DOT1L and SIRT1. The image is a representative image of three biologically independent experiments. ( f ) SIRT1 activity relative to vehicle-treated cells (dotted line). Data are from three biologically independent experiments. * P <0.05, *** P <0.001 by one-way ANOVA. ( g ) ChIP-qPCR analysis of SIRT1, PPARGC1A, GCN5 and EP300 binding on the TCF1 promoter and ( h ) TCF1 expression after siRNA transfection with indicated specific or scrambled siRNA. Data from two biologically independent experiments.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Methylation, Expressing, Co-Immunoprecipitation Assay, Activity Assay, Binding Assay

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a – c ) Inactivation of SIRT1 protects against DOT1L inhibitor-induced osteoarthritis: ( a ) C57/Bl6 wild-type mouse knees were injected with DOT1L inhibitor EPZ-5676 (5 mg kg −1 ) and SIRT1 inhibitor EX527 (1.25 mg kg –1 ), or vehicle (V) and killed after 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( a ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( b ). One experiment was performed with n =3 (vehicle), 8 (EPZ) and 10 (EPZ+EX527). * P <0.05 by one-way ANOVA. Error bars indicate mean±s.e.m. ( c ) Immunohistochemistry of TCF1 in the indicated groups. TCF1 levels are increased after EPZ treatment and normalized by additional EX527 treatment. The images are representative of three different animals. Scale bar, 200 μm. ( d , e ) Loss of DOT1L function causes severe growth retardation as demonstrated by skeletal staining ( d ) and histology of the growth plate ( e ) of 4-week-old Dot1l fl/fl ;Col2-Cre −/− (Cre-neg) and Dot1l fl/fl ;Col2-Cre +/− (Dot1l Cart-KO ) mice. ( f , g ) Increased TCF1 levels in Dot1l Cart-KO mice as shown by immunohistochemistry in the indicated mice strains in the articular cartilage ( f ) and growth plate ( g ). The images are representative of three different animals. Scale bar, 200 and 100 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Injection, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: Upon Wnt signalling activation, DOT1L-containing complexes bind Wnt target gene chromatin. DOT1L interacts with SIRT1 and inhibits its function, preventing Wnt pathway hyper-activation. When Wnt signalling is activated in the absence of DOT1L function, high SIRT1 activity mediates the recruitment of transcriptional activators to LEF1 and TCF1 genes. High Wnt signalling leads to deleterious downstream effects and loss of cartilage homeostasis.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Activation Assay, Activity Assay

Journal: Archives of toxicology

Article Title: Tributyltin induces a transcriptional response without a brite adipocyte signature in adipocyte models

doi: 10.1007/s00204-018-2268-y

Figure Lengend Snippet: Primary bone marrow cells were isolated from female, 8 week old, C57BL/6J mice, plated, and allowed to adhere for 7 days, and undifferentiated cultures were harvested at this time. In experimental cultures, the medium was replaced with basal medium supplemented with osteogenic additives, β-glycerol phosphate, ascorbate, insulin and dexamethasone. Cultures were treated with Vh (DMSO, 0.1%), Rosiglitazone (Rosi, 100 nM), LG100268 (LG268, 100 nM) or TBT (80 nM). After 4 days of culture, cells were harvested and analyzed for gene expression using microarray. The heatmaps display the significant differentially expressed (A) nuclear receptors and (B) coregulators (fdr<0.05). N = 4 independent bone marrow cultures.

Article Snippet: Rosiglitazone (Rosi) was from Cayman Chemical (Ann Arbor, MI).

Techniques: Isolation, Expressing, Microarray

Journal: Archives of toxicology

Article Title: Tributyltin induces a transcriptional response without a brite adipocyte signature in adipocyte models

doi: 10.1007/s00204-018-2268-y

Figure Lengend Snippet: Primary bone marrow cells were isolated from female, 8 week old, C57BL/6J mice, plated, and allowed to adhere for 7 days. The medium was replaced with DMEM supplemented with adipogenic additives: 10% FBS, 250 nM dexamethasone, 167 nM of 1 μg/ml human insulin, 0.5 mM IBMX. Cultures were treated with Vh (DMSO, 0.1%), Rosiglitazone (Rosi, 100 nM), LG100268 (LG268, 100 nM) or TBT (80 nM). On days 3, 5 and 7 days of differentiation, medium was replaced with adipocyte maintenance medium (DMEM, 10% FBS, 250 nM dexamethasone, 167 nM of 1 μg/ml human insulin), and the cultures were re-dosed. Following 5 or 10 days of differentiation, cells were harvested and analyzed for gene expression by RT-qPCR. (A) Common PPARγ-related genes. (B) Genes related to brite/brown adipogenesis and mitochondrial biogenesis. Data are presented as mean ± SE of 6 independently prepared bone marrow cultures. Statistically different from Vh-treated on the same day (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, ANOVA, Dunnett’s).

Article Snippet: Rosiglitazone (Rosi) was from Cayman Chemical (Ann Arbor, MI).

Techniques: Isolation, Expressing, Quantitative RT-PCR

Journal: Archives of toxicology

Article Title: Tributyltin induces a transcriptional response without a brite adipocyte signature in adipocyte models

doi: 10.1007/s00204-018-2268-y

Figure Lengend Snippet: The publicly available dataset (GSE53004) was generated from 3T3-L1 cells treated with Rosiglitazone (500 nM) or TBT (50 nM) for 10 days in DMEM with 10% FBS (Pereira-Fernandes et al. 2014). (A) GSEA analysis of curated genesets related to brown adipocyte differentiation and mitochondrial biogenesis. (B) The heatmap displays the significant differentially expressed genes (fdr<0.05) related to mitochondrial biogenesis and adipocyte browning between Rosi and TBT.

Article Snippet: Rosiglitazone (Rosi) was from Cayman Chemical (Ann Arbor, MI).

Techniques: Generated

Journal: Cancers

Article Title: The m 7 G Reader NCBP2 Promotes Pancreatic Cancer Progression by Upregulating MAPK/ERK Signaling

doi: 10.3390/cancers15225454

Figure Lengend Snippet: NCBP2 is significantly expressed in PDAC patients and correlated with poor prognosis. ( A ) Analysis of NCBP2 transcript levels in PDAC and normal tissues based on the RNA-seq data from GEPIA 2.0 cohort. ( B ) Analysis of NCBP2 transcript levels in PDAC and normal tissues based on DNA microarray data from GEO cohorts (GSE15471, GSE28735, GSE16515). ( C , D ) Overall survival and disease-free survival curves for PDAC patients with high or low NCBP2 expression in GEPIA 2.0 cohort. ( E , F ) The RNA and protein expression level of NCBP2 in HPDE and five PDAC cell lines. ( G ) The expression level of NCBP2 in PDAC and para-carcinoma tissues from tissue microarray, * p < 0.05 and *** p < 0.001.

Article Snippet: We obtained human embryonic kidney epithelial cell line 293T (HEK293T) and PDAC cell lines (Panc 05.04, PANC-1, AsPC-1, BxPC-3, MIA PaCa-2) from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: RNA Sequencing, Microarray, Expressing

Journal: Cancers

Article Title: The m 7 G Reader NCBP2 Promotes Pancreatic Cancer Progression by Upregulating MAPK/ERK Signaling

doi: 10.3390/cancers15225454

Figure Lengend Snippet: NCBP2 promotes PDAC cell growth in vitro. ( A – C ). The cell counting, 3D sphere, and colony formation assay results in control and NCBP2-knockdown Panc 05.04 and PANC-1 cells. ( D – F ). The cell counting, 3D sphere and colony formation assay results in control and NCBP2-overexpression AsPC-1 and BxPC-3 cells, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: We obtained human embryonic kidney epithelial cell line 293T (HEK293T) and PDAC cell lines (Panc 05.04, PANC-1, AsPC-1, BxPC-3, MIA PaCa-2) from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vitro, Cell Counting, Colony Assay, Control, Knockdown, Over Expression

Journal: Cancers

Article Title: The m 7 G Reader NCBP2 Promotes Pancreatic Cancer Progression by Upregulating MAPK/ERK Signaling

doi: 10.3390/cancers15225454

Figure Lengend Snippet: NCBP2 promotes PDAC cell growth in-vivo. ( A , B ) Tumor growth curves of xenograft models established from control or stable NCBP2-knockdown Panc 05.04 cells. ( C ) Assessment of tumor weight from control and NCBP2-knockdown groups. ( D , E ) The expression level of Ki-67 in tumor tissues from control and NCBP2-knockdown groups. ( F ) Body weight of nude mice in control and NCBP2-knockdown group, * p < 0.05, and ** p < 0.01.

Article Snippet: We obtained human embryonic kidney epithelial cell line 293T (HEK293T) and PDAC cell lines (Panc 05.04, PANC-1, AsPC-1, BxPC-3, MIA PaCa-2) from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vivo, Control, Knockdown, Expressing

Journal: Cancers

Article Title: The m 7 G Reader NCBP2 Promotes Pancreatic Cancer Progression by Upregulating MAPK/ERK Signaling

doi: 10.3390/cancers15225454

Figure Lengend Snippet: NCBP2 upregulates c-JUN to activate MEK/ERK signaling in a m 7 G-dependent manner. ( A ) KEGG analysis of RNA-seq data in PDAC patients with high or low NCBP2 expression from TCGA cohort. ( B ) The GSEA enrichment plot of “MAPK signaling pathway” in PDAC patients with high or low NCBP2 expression from TCGA cohort. ( C ) Immunoblotting for protein levels of total JNK/phosphorylated JNK (Thr183/Tyr185), total p38/phosphorylated p38 (Thr180/Tyr182), and total ERK/phosphorylated ERK (Thr202/Tyr204) in control and NCBP2-knockdown PDAC cells. Tubulin was used as the internal control. ( D ) Immunoblotting for protein levels of total/phosphorylated MEK and total/phosphorylated ERK (Thr202/Tyr204) in control and c-JUN-knockdown Panc 05.04 and PANC-1 cells. Tubulin was used as the internal control. ( E ) The mRNA expression levels of NCBP2 and c-JUN in control and NCBP2-knockdown Panc 05.04 and PANC-1 cells. ( F ) Immunoblotting for protein levels of NCBP2 and c-JUN in control and NCBP2-knockdown Panc 05.04 and PANC-1 cells. Tubulin was used as the internal control. ( G ) Polysome profiling results of the control and NCBP2-knockdown Panc 05.04 and PANC-1 cells. ( H ) Gene-specific m 7 G qPCR results for the m 7 G methylation levels of c-JUN in PANC 05.04 and PANC-1 cells, *** p < 0.001.

Article Snippet: We obtained human embryonic kidney epithelial cell line 293T (HEK293T) and PDAC cell lines (Panc 05.04, PANC-1, AsPC-1, BxPC-3, MIA PaCa-2) from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: RNA Sequencing, Expressing, Western Blot, Control, Knockdown, Methylation

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: ( a ) Cell proliferation and ( b ) cell viability alterations of HT-29 and SW480 colorectal cancer cell lines following different folic acid (FA) supplies. Sulforhodamine B (SRB) was used for cell proliferation detection (* p ≤ 0.05, *** p ≤ 0.001), while cell viability data were obtained by alamarBlue assay (** p ≤ 0.01). FA-depleted cells were kept in media containing 0 ng/mL FA, whereas treated cells were exposed to 100 and 10,000 ng/mL FA for 72 h. The percentages of cell proliferation and viability were given relative to samples kept in the normal growth media. FA: folic acid.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Alamar Blue Assay

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: Genomic stability detection of HT-29 and SW480 cells exposed to different folic acid (FA) concentrations (0, 100, 10,000 ng/mL). ( a ) Micronucleus (MN) scoring was performed on DAPI- and anti-γ-H2AX-stained slides. Left: We obtained the results by proportioning the cells with MN with all cells counted (** p ≤ 0.01, *** p ≤ 0.001). Right: Representative γ-H2AX-positive micronuclei are indicated with arrows. ( b ) DNA integrity was evaluated with comet assay, additionally. Left: Graphs show the changes in genomic stability in consideration of comet tail DNA percentage (* p ≤ 0.05). Right: Characteristic comets of both cell lines were captured following different treatments. FA: folic acid.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Staining, Single Cell Gel Electrophoresis

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: DNA methylation analysis of HT-29 and SW480 cell lines exposed to different folic acid (FA) concentrations. The methylation levels of long interspersed nuclear element 1 (LINE-1) CpG positions (pos 1, pos 2, pos 3) were ( a ) summarized and also ( b ) visualized individually to detect global DNA methylation changes. With the use of Reduced Representation Bisulfite Sequencing (RRBS) method, a genome-wide methylome profile of 10,000 ng/mL FA-treated cells was established in the comparison of cells kept in FA-free (0 ng/mL FA) media. ( c ) Firstly, the number of genes with altered methylation in the investigated CpG sites was assessed. “Hyper” and “hypo” sections indicate the number of genes with methylated and unmethylated CpG sites, respectively. The intersection of these two categories refers to the genes that possess both methylated and unmethylated CpG dinucleotides. ( d ) Heatmap shows the top 10 significantly ( p ≤ 0.05) enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways with the number of differentially methylated genes. ( e ) Pie charts represent the localization of differentially methylated sites (DMS) in distinct chromatin states. FA: folic acid; pos: CpG position; hyper: hypermethylation; hypo: hypomethylation; DMSs: differentially methylated sites; heterochrom/lo: heterochromatin or low signal region; txn: transcription; CNV: copy number variation; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: DNA Methylation Assay, Methylation, Methylation Sequencing, Genome Wide, Comparison

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: Genome-wide transcriptome alterations of HT-29 and SW480 cells following 10,000 ng/mL folic acid (FA) supplementation detected by Human Transcriptome Array 2.0 (HTA 2.0). ( a ) Pie charts represent the proportion of up- and downregulated genes. ( b ) Visual networks of protein–protein interactions were generated by the StringApp of Cytoscape software based on the list of genes with significant ( p ≤ 0.05) expression alterations and ≥|1.5| fold change (FC). Colors refer to the expression level of protein-coding genes (dark blue: FC ≤ −2, light blue: FC ≥ −2 and ≤−1.5, light red: FC ≥ 1.5 and ≤2, dark red: FC ≥ 2). ( c ) Top 10 genes showing significant ( p ≤ 0.05) up- and downregulation visualized with volcano plots. Gray points represent all the transcripts detected by HTA 2.0 microarray, while significantly ( p ≤ 0.05) altering genes with FC ≥ |1.5| value were marked with red and blue. P-val: p -value.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Genome Wide, Protein-Protein interactions, Generated, Software, Expressing, Microarray

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: The intersection of genome-wide DNA methylation and gene expression data obtained by Reduced Representation Bisulfite Sequencing (RRBS) and Human Transcriptome Array (HTA) 2.0 analyses. Values represent the methylome and transcriptome pattern changes of 10,000 ng/mL folic acid (FA)-treated HT-29 and SW480 cells compared to non-treated samples (0 ng/mL FA). Only genes with promoter methylation status alteration in accordance with their expression level ( p ≤ 0.05 and fold change ≥|1.5|) were listed (left) and also visualized in volcano plots (right). Gray points represent all the transcripts detected by the microarray, while blue ones highlight down- and red ones show upregulating genes from the list. met. status: DNA methylation status; met. diff.: DNA methylation difference; expr. status: gene expression status; P-val: p -value.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Genome Wide, DNA Methylation Assay, Gene Expression, Methylation Sequencing, Methylation, Expressing, Microarray

Journal: DNA research : an international journal for rapid publication of reports on genes and genomes

Article Title: Ultra-sensitive immunodetection of 5'methyl cytosine for DNA methylation analysis on oligonucleotide microarrays.

doi: 10.1093/dnares/dsi024

Figure Lengend Snippet: Figure 4. Methylation-specific PCR results. MS-PCR results for p15/CDKN2b and p16/CDKN2a after bisulfite treatment with wild-type primer (lanes 1 and 4), methylated (lanes 2 and 5) and unmethylated (lanes 3 and 6) primer sets on genomic DNA extracted from cell lines HL-60 and KG1a. m ¼ marker; amplified fragment length ¼ 150 bp.

Article Snippet: Human acute myeloid leukemia (AML) cell lines HL-60 (American Type Culture Collection) and KG1a (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ, Braunschweig, Germany) were grown in RPMI-1640 medium (Gibco Invitrogen, Communicated by Michio Oishi * To whom correspondence should be addressed.

Techniques: Methylation, Marker

Journal: DNA research : an international journal for rapid publication of reports on genes and genomes

Article Title: Ultra-sensitive immunodetection of 5'methyl cytosine for DNA methylation analysis on oligonucleotide microarrays.

doi: 10.1093/dnares/dsi024

Figure Lengend Snippet: Figure 5. Microarray methyl-cytosine immunodetection on genomic DNA. Hybridization of restriction enzyme digested genomic DNA of two AML tumor cell lines. A. Top scan of HL-60 (1) and bottom scan of KG1a (2) microarray. Overlaid scans display methylation signal (cyanine 3: green signal) of KG1a DNA compared to HL-60 and capture oligonucleotide scan (cyanine 5: red signal). For negative control () spotted nonsense oligonucleotide without 50mC were used; positive control (þ) with 50mC modified control oligonuc- leotide. Original magnification: ·100; spot distance: 150 mm; pixel resolution: 0.516 mm. B. Box plot analysis of A reveal that HL-60 is methylation negative for p15/CDKN2b and p16/CDKN2a but methylation positive for E-cadherin. Results for KG1a are inverted. Box plots of 45 individual spots per gene show median, 75% percent- ile and outliers. P < 0.01 for all three genes.

Article Snippet: Human acute myeloid leukemia (AML) cell lines HL-60 (American Type Culture Collection) and KG1a (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ, Braunschweig, Germany) were grown in RPMI-1640 medium (Gibco Invitrogen, Communicated by Michio Oishi * To whom correspondence should be addressed.

Techniques: Microarray, Immunodetection, DNA Hybridization, Methylation, Negative Control, Positive Control, Control

Journal: American Journal of Cancer Research

Article Title: ELOVL2: a novel tumor suppressor attenuating tamoxifen resistance in breast cancer

doi:

Figure Lengend Snippet: Optimization of plasmid and siRNA transfection into cultured cells. MCF-7/TamR cells are transiently transfected with an ORF to induce upregulation or with a siRNA to induce downregulation of the indicated gene. A. Overexpression of ELOVL2. Recombinant plasmid DNA (1-2 μg/mL) is transfected, and 2 μg/mL is used for further transfection. B. Expression of ELOVL2 is confirmed by qPCR and Western blot analysis. C. Downregulation of THEM4 using siRNA, as judged by qPCR. Two siRNAs targeting different sites of THEM4 were used in MCF-7/TamR cells (left) and MCF-7/TamR ELOVL2 ORF cells (right).

Article Snippet: ELOVL2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_017770","term_id":"1519313823","term_text":"NM_017770"}} NM_017770 ) Human ORF Clone Lenti Particle (Cat. No. RC209232L4V) , , , , Origene.

Techniques: Plasmid Preparation, Transfection, Cell Culture, Over Expression, Recombinant, Expressing, Western Blot

Journal: American Journal of Cancer Research

Article Title: ELOVL2: a novel tumor suppressor attenuating tamoxifen resistance in breast cancer

doi:

Figure Lengend Snippet: Information of primers for qPCR and siRNAs employed in this study

Article Snippet: ELOVL2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_017770","term_id":"1519313823","term_text":"NM_017770"}} NM_017770 ) Human ORF Clone Lenti Particle (Cat. No. RC209232L4V) , , , , Origene.

Techniques: Sequencing, Negative Control, Plasmid Preparation

Journal: American Journal of Cancer Research

Article Title: ELOVL2: a novel tumor suppressor attenuating tamoxifen resistance in breast cancer

doi:

Figure Lengend Snippet: Top 20 genes of which methylation are highly altered in the MCF-7/TamR cells

Article Snippet: ELOVL2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_017770","term_id":"1519313823","term_text":"NM_017770"}} NM_017770 ) Human ORF Clone Lenti Particle (Cat. No. RC209232L4V) , , , , Origene.

Techniques: Methylation

Journal: American Journal of Cancer Research

Article Title: ELOVL2: a novel tumor suppressor attenuating tamoxifen resistance in breast cancer

doi:

Figure Lengend Snippet: ELOVL2 is hypermethylated and downregulated in TamR breast cancer. A. Downregulation of ELOVL2 in MCF-7/TamR. Expression of ELOVL2 is examined by Western blot analysis. B. Demethylation of CpGs is induced by Aza in the MCF-7/TamR cells and ELOVL2 expression is analyzed by qPCR. C. Hypermethylation and downregulation of ELOVL2 in breast cancer tissues. MSP and qPCR are performed for breast tissues from Tam-sensitive and Tam-resistant cancer patients. N: number of samples. D. Immunohistochemical analysis of ELOVL2 in Tam-sensitive and Tam-resistant cancer tissues. Three tissue sets are analyzed and the protein expression is denoted by the bar graph. Images from two tissue sets are represented. Scale bar, 50 μm. E. Kaplan-Meier survival analysis of ELOVL2 expression in breast cancer. Samples (n = 1,746) are stratified into two groups based on ELOVL2 expression level. The log-rank test is performed in all tumor samples using distant metastasis-free survival (DMFS) as the endpoint. High ELOVL2 expression is significantly associated with higher DMFS in cancer patients (P < 0.005).

Article Snippet: ELOVL2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_017770","term_id":"1519313823","term_text":"NM_017770"}} NM_017770 ) Human ORF Clone Lenti Particle (Cat. No. RC209232L4V) , , , , Origene.

Techniques: Expressing, Western Blot, Immunohistochemical staining

Journal: American Journal of Cancer Research

Article Title: ELOVL2: a novel tumor suppressor attenuating tamoxifen resistance in breast cancer

doi:

Figure Lengend Snippet: TamR cancer cells show a lowered ELOVL2 expression and grow faster in xenograft tumor tissue. (A) ELOVL2 is upregulated in MCF-7/TamR by transiently transfecting a recombinant plasmid vector. Effect of ELOVL2 on cell proliferation is examined by CCK assay. (B) MCF-7/TamR cells grow faster than MCF-7 in a xenograft animal model. MCF-7 and MCF-7/TamR cells are subcutaneously injected into nude mice and the tumor volume is measured for 7 weeks. n = 8. (C) Mice are sacrificed 8 weeks after transplantation to obtain the tumor tissues. Expression of ELOVL2 in the xenografted tumor is examined by Western blot analysis (D) and immunohistochemical analysis (E). Three tumor sets are analyzed and the average protein expression is denoted in a bar graph. Representative images are shown. Scale bar, 50 μm.

Article Snippet: ELOVL2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_017770","term_id":"1519313823","term_text":"NM_017770"}} NM_017770 ) Human ORF Clone Lenti Particle (Cat. No. RC209232L4V) , , , , Origene.

Techniques: Expressing, Recombinant, Plasmid Preparation, Animal Model, Injection, Transplantation Assay, Western Blot, Immunohistochemical staining

Journal: American Journal of Cancer Research

Article Title: ELOVL2: a novel tumor suppressor attenuating tamoxifen resistance in breast cancer

doi:

Figure Lengend Snippet: ELOVL2 suppresses tumor growth and recovers Tam sensitivity in in vitro and in vivo animal model. A. Effect of ELOVL2 on Tam sensitivity is examined after treating the ELOVL2 ORF-transfected MCF-7/TamR cells with Tam and then measuring the growth rate by CCK assay. B. Effect of ELOVL2 on Tam sensitivity is examined by colony formation assay. All the assays are performed in triplicates, and the result is depicted as mean ± SE. Representative images are shown for the colony formation assay. NC, negative control vector. ORF, open reading frame. C. MCF-7/TamR cells that are stably transfected with ELOVL2-expressing cDNA or control DNA are subcutaneously injected into nude mice and Tam is administered 3 weeks after cell injection. The tumor volume is measured for 7 weeks. D. At week 8, mice are sacrificed to obtain the tumor tissues (n = 6 for corn oil-treated mice; n = 4 for Tam-treated mice).

Article Snippet: ELOVL2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_017770","term_id":"1519313823","term_text":"NM_017770"}} NM_017770 ) Human ORF Clone Lenti Particle (Cat. No. RC209232L4V) , , , , Origene.

Techniques: In Vitro, In Vivo, Animal Model, Transfection, Colony Assay, Negative Control, Plasmid Preparation, Stable Transfection, Expressing, Injection

Journal: American Journal of Cancer Research

Article Title: ELOVL2: a novel tumor suppressor attenuating tamoxifen resistance in breast cancer

doi:

Figure Lengend Snippet: Highest confidence network of genes displaying altered ELOVL2 expression in MCF-7/TamR. ELOVL2 is overexpressed in MCF-7/TamR and a genome-wide expression analysis is performed. (A) Heatmap analysis of 969 genes that are significantly deregulated by ELOVL2. The data are from the microarray in duplicates. (B) Highest confidence network of genes displaying altered expression identifies “Cardiovascular Disease, Cell-To-Cell Signaling and Interaction, Inflammatory Response” pathway and “Behavior, Reproductive System Development and Function, Cardiac Infarction” pathway as the top networks. Genes that are upregulated are shaded in red, whereas those that are downregulated are shaded in green, with the color intensity signifying the magnitude of expression change. Solid lines representing direct interactions, and dashed lines representing indirect interactions. (C) Top 10 canonical pathways and (D) disease and function annotation of the genes of which expression is significantly altered by ELOVL2. The most significant canonical pathway is “Antigen Presentation Pathway” and disease and function annotation is “Solid tumor”.

Article Snippet: ELOVL2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_017770","term_id":"1519313823","term_text":"NM_017770"}} NM_017770 ) Human ORF Clone Lenti Particle (Cat. No. RC209232L4V) , , , , Origene.

Techniques: Expressing, Genome Wide, Microarray

Journal: American Journal of Cancer Research

Article Title: ELOVL2: a novel tumor suppressor attenuating tamoxifen resistance in breast cancer

doi:

Figure Lengend Snippet: ELOVL2 regulates genes in AKT and ERa networks. A few genes in the top IPA network (Figure 7B) are selected and qPCR is performed to confirm the microarray expression data. All genes show the same expression trend with being up- or down-regulated in ELOVL2 overexpression cells compared with the MCF-7/TamR cells, although the alteration level is not the same.

Article Snippet: ELOVL2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_017770","term_id":"1519313823","term_text":"NM_017770"}} NM_017770 ) Human ORF Clone Lenti Particle (Cat. No. RC209232L4V) , , , , Origene.

Techniques: Microarray, Expressing, Over Expression

Journal: American Journal of Cancer Research

Article Title: ELOVL2: a novel tumor suppressor attenuating tamoxifen resistance in breast cancer

doi:

Figure Lengend Snippet: THEM4 is downregulated by ELOVL2 and increases Tam resistance. (A) Increased expression of THEM4 in MCF-7/TamR cells. qPCR (left) and Western blot analysis (right) are performed in MCF-7/TamR and MCF-7 cells. (B) Downregulation of THEM4 by ELOVL2 in MCF-7/TamR cells, determined by qPCR. ELOVL2 ORF: cells stably transfected with ELOVL2 cDNA; ORF NC; negative cDNA control. (C) Increased expression of THEM4 in the xenografted MCF-7/TamR but suppression by ELOVL2. Western blot analysis is performed for tumor tissues from ELOVL2 ORF and control. Immunohistochemical analysis of THEM4 in xenografted tumor tissue of MCF-7/TamR (D) and cells stably transfected with ELOVL2 cDNA (E) Scale bar, 50 μm. Effect of THEM4 on recovery of Tam sensitivity. THEM4 is downregulated via siRNA #2 at a final concentration of 40 nM in MCF-7/TamR (F) and ELOVL2-overexpressing MCF-7/TamR cells (G). Sensitivity to Tam is examined by colony formation assay. Representative images from three independent assays are shown. (H) Effect of THEM4 on cell proliferation is examined by a dye-based CCK assay. (I) Effect of THEM4 on TamR is examined by exposing the cells to Tam after downregulating the gene with siRNA. All the assays are performed in triplicates, and the result is depicted as mean ± SE.

Article Snippet: ELOVL2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_017770","term_id":"1519313823","term_text":"NM_017770"}} NM_017770 ) Human ORF Clone Lenti Particle (Cat. No. RC209232L4V) , , , , Origene.

Techniques: Expressing, Western Blot, Stable Transfection, Transfection, Immunohistochemical staining, Concentration Assay, Colony Assay

Journal: American Journal of Cancer Research

Article Title: ELOVL2: a novel tumor suppressor attenuating tamoxifen resistance in breast cancer

doi:

Figure Lengend Snippet: Schematic illustration of the regulatory pathway by ELOVL2. The uptake ratio of Tam across plasma membrane in MCF-7/TamR cells is similar to that in the parental MCF-7 cells. ERa and ELOVL2 crosstalk to regulate each other. ELOVL2 blocks the PI3K/AKT/mTOR pathway via inhibiting THEM4 and PI3K. In TamR cancer, ELOVL2 is downregulated by hypermethylation, resulting in loss of inactivation of AKT and also downstream genes such as CREB and mTOR, or activation of downstream genes such as BMF, eventually leading to Tam resistance and increased cell proliferation.

Article Snippet: ELOVL2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_017770","term_id":"1519313823","term_text":"NM_017770"}} NM_017770 ) Human ORF Clone Lenti Particle (Cat. No. RC209232L4V) , , , , Origene.

Techniques: Activation Assay

Journal: American Journal of Cancer Research

Article Title: ELOVL2: a novel tumor suppressor attenuating tamoxifen resistance in breast cancer

doi:

Figure Lengend Snippet: ELOVL2 downregulates total amount of AKT and p-AKT. A. Downregulation of genes in the AKT pathway by ELOVL2. qPCR is performed for seven genes in the AKT pathway in the ELOVL2-overexpressing MCF-7/TamR cells. Samples are analyzed in triplicates, and the result is shown as mean ± SE. B. Protein level of AKT and p-AKT is analyzed in the MCF-7/TamR cells transiently transfected with a recombinant ELOVL2 cDNA plasmid. Western blot analysis is performed in triplicates, and a representative image is shown with a bar graph depicted as mean ± SE.

Article Snippet: ELOVL2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_017770","term_id":"1519313823","term_text":"NM_017770"}} NM_017770 ) Human ORF Clone Lenti Particle (Cat. No. RC209232L4V) , , , , Origene.

Techniques: Transfection, Recombinant, Plasmid Preparation, Western Blot

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 2 DACH1 deletion PCa enhances AR signaling. A Interrogation of human PCa gene expression data [26], showing candidate genetic drivers ERG, ETV1/ETV4/FLI1, SPOP, FOXA1, and unknown. Samples with DACH1 homozygous (deep) genetic deletions (29/333) are shown as an additional subtype. The AR score (the average of the AR target gene expression) refers to a group of AR-responsive genes [26], and together with the expression Z-score of the AR target genes, are shown as colorimetric scales. The AR score-based gene names are shown. The androgen receptor (AR) activity, inferred by the induction of AR target genes, was increased in DACH1 homozygous (‘deep’) deletion PCa compared with normal (P = 2 × 10−5 by t-test) and ERG mutation groups (P = 0.003 by t-test). B AR mRNA and AR protein levels, shown for each DACH1 deletion sample, were not significantly different. C The iCluster [29], mRNA cluster, and SCNA (somatic copy-number alteration), and DNA methylation status are shown for the PCa classified by the corresponding gene deletion subtypes. D DACH1 homozygous deletions were enriched for iCluster 2 and 3 [29], mRNA cluster 2 (P = 0.0003 by Fisher exact test, SCNA (“more” somatic copy-number alteration, P = 0.0004 by Fisher exact test), but not for DNA methylation.

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Gene Expression, Targeted Gene Expression, Expressing, Activity Assay, Mutagenesis, DNA Methylation Assay

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 3 Prostate-specific Dach1 gene deletion promotes prostate hyperplasia and dysplasia in OncoMice (15 weeks). A Schematic representation of transgenes integrated into mice. B Representative immunohistochemistry for Dach1, with data quantitated as mean ± standard error of the mean (SEM) for N = 20 (4 separate mice, with 5 views per mouse, in each group). C Blinded quantitative histology grading of prostate of multigenic mice at 15 weeks. Data are shown as mean ± SEM for N = 15 (5 separate mice, with 3 prostate areas [anterior, ventral, lateral] per mouse) in each group). H&E staining demonstrates the presence of a focal atypical intraductal proliferation in Dach1−/−prostate, compatible with prostatic intraepithelial neoplasia (PIN). Representative immunohistochemistry with results shown as mean ± SEM for Ki-67 (n = 20, 4 separate mice for each genotype, 5 views per mouse) (D), Beclin 1 (n = 9, 3 separate mice for each genotype, 3 views per mouse) (E); and AR (n = 15 for Dach1wt/wt mice, 3 separate mice, 5 views per mouse) (n = 12 for Dach1fl/flmice, 3 separate mice, 2 views for one mouse and 5 views for other two mice) (F). Scale bars, 50 μm. A Student’s t test was performed for all comparisons.

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Immunohistochemistry, Staining

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 4 Prostate-specific Dach1 gene deletion in TRAMP mice induces PIN lesions with increased TGFβ activity. Genome-wide expression analysis of TRAMP Dach1+/+ vs. Dach1−/−PIN lesions was analyzed for enrichment of known targets of upstream regulators using Ingenuity Pathway Analysis (IPA) and represented as (A) barplot was calculated by IPA activation Z-score labeled and as (B) bubble plot with size of the bubbles proportional to –log10 p values. C IHC was conducted for SMAD activation using SMAD2P, quantitated and shown as (D) mean ± SEM (n = 15 for Dach1wt/wt mice, 3 separate mice, 5 views per mouse) (n = 10 for Dach1fl/flmice, 2 separate mice, 5 views per mouse). E–G Western blot of either PCa cell lines for the presence of DACH1 (E, F) or (G) TGFβ-treated (10 ng/ml for 24 h) PC3 cells illustrating induction of nuclear vimentin and cytoplasmic cyclin D1. Protein loading controls are β-tubulin (a marker of cytoplasmic proteins) and Lamin B1 (a marker for nuclear protein enrichment). H Microarray-based gene expression analysis of PC3 cells stably expressing DACH1, showing restraint of genes mediating TGFβ signaling (shown with blue arrows), including reduction of TGFB2 and TGFBR2 [33].

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Activity Assay, Genome Wide, Expressing, Activation Assay, Labeling, Western Blot, Marker, Protein Enrichment, Microarray, Gene Expression, Stable Transfection

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 6 DACH1 facilitates the recruitment of, and co-accumulates with, Ku70/Ku80 proteins at sites of DNA damage. A Co-accumulation of Ku-70/Ku-80 at laser micro irradiation-induced DSBs sites in Dach1+/+ 3T3 cells. B, C 24 h after transfection, the accumulation of DACH1 and Ku70/Ku80 in Dach1−/−3T3 cells transfected with EGFP or EGFP-tagged DACH1 and red fluorescent protein (RFP)-tagged Ku70 or RFP-tagged Ku80 expression vectors were treated with laser micro-irradiation (403 nm) to induce DSBs. Time is shown after micro-irradiation. Accumulation of the transfected proteins was indicated by EGFP (green) or RFP (red) fluorescence at laser-irradiated sites. Co-accumulation was visualized in yellow merged images. Time is shown in minutes and -fold increase in foci intensity is shown as mean ± SEM for N = 5 separate cells.

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Irradiation, Transfection, Expressing