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: 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: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Molecular events for CRBP1 gene in cervical epithelium samples. A: In order to know the gain of copy number of the CRBP1 gene, DNA of healthy cervix and CC samples, were subjected to real time PCR with specific Taqman probes. White bar (healthy cervix samples) represents the mean of the normal cervices (n = 26) without extra copies of CRBP1 gene. Black bars show CC samples with gain of copy number (2-20X); while gray dotted line bars are showing CC samples that do not change in the copies number. Values above the cut-off line (as 1), being assigned as increased gene copy number compared with normal cervical epithelium. CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [26]. In X-axis represents cervical samples, Y-axis relative copies fold change of CRBP1 gene. B: CRBP1 expression was observed as positive immunostaining result on tissue microarray as mentioned in Methods section The DNAs used for gain of copy number (panel A) were also used for the methylation assay. Methylation result represents the methylation of the CRBP1 promoter. In this case, each healthy or CC sample, correspond to each column for CRBP1 expression and methylation status. Interestingly, in most of the cases, there was an association between the lack of expression of the CRBP1 gene and its methylation status.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunostaining, Microarray, Methylation

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: CRBP1 immunodetection in the uterine cervix samples. A: (1) Cytoplasmic CRBP1 expression is present in cells of the basal layer of normal cervical epithelium (healthy tissue); (2) the immunodetection in the transformed cells of a cervical cancer (CC03) tissue harboring gain of CRBP1 gene. (3) CC samples without gain CRBP1 gene showing negative immunostaining (CC16 sample). A kidney tissue section (4) was used as positive control, while a heart tissue section for negative control (5). B: cervical progression spectrum. The tissue section shows a brownish reaction (positive reaction) in the basal cell layer of the “normal” region, in the high-grade lesion, and also in the invasive region. All tissue sections were hematoxylin counterstained, 200X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunodetection, Expressing, Transformation Assay, Immunostaining, Positive Control, Negative Control, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Association between CRBP1 gene gain copy number and its expression in cervical cancer samples

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Immunodetection

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Correlation between CRBP1 expression and clinic pathological variables in cervical cancer

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Activity Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Immunolocalization of CRBP1 by immunofluorescence in cervical cells. Nuclei were Dapi stained in blue color (A-C). The immunodetection of CRBP1 was observed in green color (D-F). Cytoplasmic immunodetection of CRBP1 in the merge imaging (G-I). 100X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunofluorescence, Staining, Immunodetection, Imaging, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Methylation promoter of CRBP1 gene in cervical cancer samples. Example of CRBP1 gene promoter methylation analysis. Lanes: Healthy cervix sample, CC03 and CC06 samples with un-methylated status; lanes CC 10 and CC 16 with methylated status; HeLa cells as un-methylated control (109 bp), or MCF-7 cells as methylated control (99 bp). MW: molecular weight marker of 100 bp.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Methylation, Molecular Weight, Marker

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: BMC Medical Genomics

Article Title: Association of differential gene expression with imatinib mesylate and omacetaxine mepesuccinate toxicity in lymphoblastoid cell lines

doi: 10.1186/1755-8794-5-37

Figure Lengend Snippet: Experimental and analytical protocol used in the study. Experimental series 1 was undertaken to estimate the differential gene expression in 48,803 probes upon treatment with imatinib or omacetaxine. For these experiments both drugs were administered at a dose of IC20. Experimental series 2 was undertaken to measure an individual’s sensitivity to drug response (SDR) which was estimated as the slope of the regression line (arrows) between log of dose administered and cell viability. Data from these experiments were also used to estimate the IC20 values used in Experimental Series 1. Differential gene expression (iFC and oFC) was tested for statistical significance for departure from zero as well as for association with the corresponding SDR as shown. Details of the statistical methods mentioned in the figure are provided in the text.

Article Snippet: Imatinib mesylate (LKT Laboratories, St Paul, MN) and omacetaxine mepesuccinate (ChemGenex, Melbourne, Australia) were solubilized in water at 10 mM and 10 μM concentrations, respectively, and stored at −20°C until used in experiments.

Techniques: Gene Expression

Journal: BMC Medical Genomics

Article Title: Association of differential gene expression with imatinib mesylate and omacetaxine mepesuccinate toxicity in lymphoblastoid cell lines

doi: 10.1186/1755-8794-5-37

Figure Lengend Snippet: Heritability of the inhibitory concentration of experimental drugs

Article Snippet: Imatinib mesylate (LKT Laboratories, St Paul, MN) and omacetaxine mepesuccinate (ChemGenex, Melbourne, Australia) were solubilized in water at 10 mM and 10 μM concentrations, respectively, and stored at −20°C until used in experiments.

Techniques: Concentration Assay

Journal: BMC Medical Genomics

Article Title: Association of differential gene expression with imatinib mesylate and omacetaxine mepesuccinate toxicity in lymphoblastoid cell lines

doi: 10.1186/1755-8794-5-37

Figure Lengend Snippet: Differential gene expression response upon treatment with imatinib or omacetaxine. (A-B) Volcano plots depicting the extent (x-axis) and significance (y-axis) of differential gene expression for each probe set (n = 48,803) after treatment with either imatinib (A) or omacetaxine (B). (C) Results of k-means clustering of the statistically significant probes based on their differential gene expression in response to imatinib (x-axis) or omacetaxine (y-axis) treatment. The color codes for each cluster are indicated in the index, clusters have been referenced consistently throughout the manuscript. The lack of data towards the center of the scatter plot is due to probes that were not significant (in response to either imatinib or omacetaxine treatment), which are not shown in this figure. (D) Point estimates and 95% confidence ellipses for the association of differential gene expression with sensitivity of drug response (SDR). Plotted in this chart are the regression coefficients from the polygenic models (equation (2)) for the set of probes belonging to the color-coded clusters identified in panel C. (E) Heritability of differential gene expression. The data are shown separately for probe sets belonging to each cluster identified in panel C. Pie charts at the top demonstrate the proportion of probe sets within the corresponding cluster that showed a heritability value exceeding zero for differential gene expression in response to imatinib as well as omacetaxine. The bar charts show the mean heritability of imatinib (blue) and omacetaxine (red) response for genes within each cluster. For reference, color-coded background is shown on the chart corresponding to the clusters. Within each cluster, the difference in the mean heritability for imatinib and omacetaxine response was assessed using a paired Student’s t test, the result of which is shown as the p-value at the top of the bars. ih2r, heritability of differential gene expression in response to imatinib; oh2r, heritability of differential gene expression in response to omacetaxine.

Article Snippet: Imatinib mesylate (LKT Laboratories, St Paul, MN) and omacetaxine mepesuccinate (ChemGenex, Melbourne, Australia) were solubilized in water at 10 mM and 10 μM concentrations, respectively, and stored at −20°C until used in experiments.

Techniques: Gene Expression

Journal: BMC Medical Genomics

Article Title: Association of differential gene expression with imatinib mesylate and omacetaxine mepesuccinate toxicity in lymphoblastoid cell lines

doi: 10.1186/1755-8794-5-37

Figure Lengend Snippet: Validation of the microarray results by qPCR. (A) Scatter plot of the differential gene expression in response to imatinib (blue) and omacetaxine (red). Both axes show log-transformed differential gene expression. Results are for all five genes that were assayed by microarray and qPCR. Since qPCR used HPRT1 as the normalizer gene, the microarray based results are shown after correction for HPRT1 expression. (B) Mean differential gene expression for the five selected genes by qPCR (left panel) and microarray (right panel) in response to imatinib (blue bars) and omacetaxine (red bars) treatment. The bars represent log-transformed differential gene expression values.

Article Snippet: Imatinib mesylate (LKT Laboratories, St Paul, MN) and omacetaxine mepesuccinate (ChemGenex, Melbourne, Australia) were solubilized in water at 10 mM and 10 μM concentrations, respectively, and stored at −20°C until used in experiments.

Techniques: Biomarker Discovery, Microarray, Gene Expression, Transformation Assay, Expressing

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: PLoS ONE

Article Title: The Effects of Different Repetitive Transcranial Magnetic Stimulation (rTMS) Protocols on Cortical Gene Expression in a Rat Model of Cerebral Ischemic-Reperfusion Injury

doi: 10.1371/journal.pone.0139892

Figure Lengend Snippet: Fold-changes in mRNAs (RQs) of all genes that showed expression on the microarray in MCAO and ShMCAO group using the control group as a calibrator. C and Sh in the group significance column indicate significant difference to CON and ShMCAO group, respectively.

Article Snippet: Gria1 , Glutamate receptor ionotropic AMPA 1 , Rn00709588_m1 , 1.07 , , 0.94 , 0.305 , 0.401 , 2 , 13.553 , 0.677.

Techniques: Expressing, Microarray, Control, Binding Assay, Membrane

Journal: PLoS ONE

Article Title: The Effects of Different Repetitive Transcranial Magnetic Stimulation (rTMS) Protocols on Cortical Gene Expression in a Rat Model of Cerebral Ischemic-Reperfusion Injury

doi: 10.1371/journal.pone.0139892

Figure Lengend Snippet: Fold-changes in mRNAs (RQs) of all genes that showed expression on the microarray after two weeks of rTMS using the MCAO group as a calibrator. MCAO, 1, 5, and Sh in the group significance column indicate significant difference to MCAO, 1Hz, 5 Hz, and ShSTIM groups, respectively.

Article Snippet: Gria1 , Glutamate receptor ionotropic AMPA 1 , Rn00709588_m1 , 1.07 , , 0.94 , 0.305 , 0.401 , 2 , 13.553 , 0.677.

Techniques: Expressing, Microarray