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: Stem cell research & therapy

Article Title: Sox9 transcriptionally regulates Wnt signaling in intestinal epithelial stem cells in hypomethylated crypts in the diabetic state.

doi: 10.1186/s13287-017-0507-4

Figure Lengend Snippet: Fig. 2 Expression and promoter methylation of DNA methyltransferases in intestinal epithelium. a, b Decreased Dnmt1 expression is detected in db/db crypts and intestinal epithelium stem cells (IESCs) by qRT-PCR using the FACS cell population (a) and Western blot using the MACS cell population (b left) and quantification of immunoblot bands from Western blot (b right). mRNA and protein levels are expressed relative to β-actin. Mean ± SE; n ≥6; *P < 0.05, **P < 0.01 by t test. C represents the control db/+ group; D represents the diabetic db/db group. c Promoter methylation microarray of Dnmt3b indicates promoter hypermethylation not only in db/db mice (orange bars) but also in db/+ mice (light blue bars). Values in brackets indicate the peak score that reflects the probability of positive enrichment and average P value scores. d, e Validation of Dnmt3b microarray results by bisulfite sequence. The table indicates the results of bisulfite sequencing analyses of 10 bacterial colonies per group (d). The CpG sites validated are the sites that exhibit a peak signal in the microarray. Black circles represent methylated CpG sites; white hollow circles represent unmethylated CpG sites; crosses represents methylation status not determined . Bar graph showing the ratio of methylated cyto- sine in CpG within the regions analyzed (e). f, g Immunohistochemical staining of Dnmt1 (f) and Dnmt3a (g). Scale bars = 50 μm. Diff differenti- ated cells, N.S. not significant

Article Snippet: The membranes were probed overnight at 4 °C with the following primary antibodies: rabbit anti-mouse Dnmt1 antibody (1:2000; Abcam, Cambridge, MA, UK), rabbit anti-mouse Dnmt3a antibody (1:1000; Abcam), rabbit anti-mouse Sox9 antibody (1:1500; Abcam), and rabbit anti-mouse β-actin antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Methylation, Quantitative RT-PCR, Western Blot, Control, Microarray, Biomarker Discovery, Sequencing, Methylation Sequencing, Immunohistochemical staining, Staining

Journal: Stem cell research & therapy

Article Title: Sox9 transcriptionally regulates Wnt signaling in intestinal epithelial stem cells in hypomethylated crypts in the diabetic state.

doi: 10.1186/s13287-017-0507-4

Figure Lengend Snippet: Fig. 3 Promoter methylation levels and gene expression of Wnt signaling-related genes. a (left) Hierarchical clustering analysis on the basis of the DMEP-related genes related with Wnt signaling. Each row indicates a DMEP-related gene, and the corresponding gene name is indicated on the right. Each column indicates a sample analyzed. Methylation levels range from unmethylated (green) to fully methylated (red), as indicated by the color legend at the top of the graph. a (right) The degree of methylation of Wnt signaling pathway-related genes among DMEP-related genes. The bar height reflects the degree of methylation. b, c The mRNA levels of Wnt signaling-related genes in crypts and intestinal epithelium stem cells (IESCs) using the FACS cell population. d (upper) Western blot analysis of Sox9 using the MACS cell population, and d (lower) quantification of immunoblot bands from three repetitions of western blot experiments. e Immunohistochemical staining of Sox9 in the small intestinal epithelium. The arrows indicate cells expressing low levels of Sox9, which were considered IESCs. Scale bars = 50 μm. mRNA and protein levels are expressed relative to β-actin. Mean ± SE; n ≥6 (mRNA); n ≥3 (protein); *P < 0.05, **P < 0.01, ***P < 0.001 by t test. N.S. not significant

Article Snippet: The membranes were probed overnight at 4 °C with the following primary antibodies: rabbit anti-mouse Dnmt1 antibody (1:2000; Abcam, Cambridge, MA, UK), rabbit anti-mouse Dnmt3a antibody (1:1000; Abcam), rabbit anti-mouse Sox9 antibody (1:1500; Abcam), and rabbit anti-mouse β-actin antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Methylation, Gene Expression, Western Blot, Immunohistochemical staining, Staining, Expressing

Journal: Cell Reports

Article Title: Aryl Hydrocarbon Receptor Contributes to the Transcriptional Program of IL-10-Producing Regulatory B Cells

doi: 10.1016/j.celrep.2019.10.018

Figure Lengend Snippet:

Article Snippet: Negative CD43- Isolation Kit , Miltenyi Biotec , Cat# 130-049-801.

Techniques: Western Blot, Recombinant, Methylation, Adjuvant, Enzyme-linked Immunosorbent Assay, Isolation, cDNA Synthesis, SYBR Green Assay, DNA Library Preparation, Purification, Bicinchoninic Acid Protein Assay, Microarray, Ex Vivo, Generated, Software, Red Blood Cell Lysis, Staining, Lysis

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: 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: 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: Nature Communications

Article Title: The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation

doi: 10.1038/ncomms7537

Figure Lengend Snippet: ( a ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression by 24-h control, IFN-γ- and/or TNF-α-stimulated undifferentiated KCs or HPV16+KCs. Gene expression was normalized using GAPDH as the calibrator gene. Fold changes over control-stimulated undifferentiated KCs were calculated and depicted. ( b ) Enzyme-linked immunosorbent assay for CCL2, RANTES, IL-8 and CXCL9 in cleared supernatants of 24-h control, IFN-γ- and/or TNF-α-stimulated undifferentiated KCs or HPV16+KCs. ( c ) PBMCs migration towards cleared supernatants of 24-h control, IFN-γ- and/or TNF-α-stimulated KCs or HPV16+KCs. A representative example of three different donors is shown. ( d ) RelA phosphorylation, acetylation and total levels in KCs and HPV16+KCs stimulated with TNF-α for 0, 5, 15 and 30 min. ( e ) RelA acetylation and total levels at steady state in three human primary KC donor pools originating from human foreskin keratinocytes (HFKs), human vaginal keratinocytes (HVKs) or human cervical keratinocytes (HCKs) and two HPV16+genome-transfected primary KC pools of foreskin (HFK16) or vaginal (HVK16) origin. ( f ) RT–qPCR of CCL2, RANTES, IL-8 and CXCL9 in HPV16+KCs and KCs. Gene expression was normalized using GAPDH as the calibrator gene. Gene expression in HPV16+KCs was standardized over KCs. All data are representative for at least three independent experiments. Error bars indicate s.d. P values were determined using Welch-corrected unpaired t -tests. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Immunodetection was achieved with anti-p65 (1:1,000, sc-372, Santa Cruz), anti-phospho-p65 (Ser536; 1:1,000, #3033 Cell Signaling Technology (CST)), anti-acetyl-p65 (Lys310; 1:1,000, #3045 CST), anti-IFRD1 (1:400, T2576 Sigma-Aldrich), β-actin (1:10,000, Sigma-Aldrich) primary antibodies, and horseradish peroxidase-coupled anti-mouse (1:5,000; CST) and horseradish peroxidase-coupled anti-rabbit (1:5,000, CST) secondary antibodies.

Techniques: Quantitative RT-PCR, Expressing, Control, Gene Expression, Enzyme-linked Immunosorbent Assay, Migration, Phospho-proteomics, Transfection

Journal: Nature Communications

Article Title: The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation

doi: 10.1038/ncomms7537

Figure Lengend Snippet: Microarray intensities for ( a ) all KATs , HDACs and SIRTs , and ( b ) IFRD1 in four independent KCs and four independent hrHPV+KCs represented in a box plot. The box contains the 1st quartile up to the 3rd quartile; the median is represented as a line; whiskers represent the values of the outer two quartiles. ( c ) IFRD1 mRNA expression of one representative control primary KC culture and two HPV16+KC cultures (left panel), in HFK16 cells transfected with siControl or siHPV16 (middle panel) and in primary KCs that are either mock infected or infected with native HPV16 virions (right panel), as measured by RT–qPCR. ( d ) IFRD1 protein expression in three human primary keratinocyte (KC) donor pools originating from human foreskin keratinocytes (HFKs), human vaginal keratinocytes (HVKs) or human cervical keratinocytes (HCKs) and two HPV16+genome-transfected primary KC pools of foreskin (HFK16) or vaginal (HVK16) origin (left panel) in HFK16 cells transfected with siControl or siHPV16 (middle panel) and in primary KCs that are either mock infected or infected with native HPV16 virions (right panel), as measured by western blot. ( e ) Immunohistochemical staining for IFRD1, HPV16 E2, p16 and negative antibody control of a vulvar intraepithelial neoplasia (VIN) lesion, one representative donor of two shown. Counterstaining was done using haematoxylin. Scale bar, 500 μm. ( f ) IFRD1, RelA K310 acetylation and total RelA levels in 24-h non- or IFN-γ- and TNF-α-stimulated control or IFRD1 knockdown (KD) HPV16+KCs. ( g ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in steady-state control or IFRD1 KD HPV16+KCs. ( h ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and/or TNF-α-stimulated control or IFRD1 KD HPV16+KCs. ( i ) Enzyme-linked immunosorbent assay for CCL2, RANTES, IL-8 and CXCL9 in cleared supernatants of 24-h non- or IFN-γ- and/or TNF-α-stimulated control or IFRD1 KD HPV16+KCs. ( j ) PBMCs migration towards cleared supernatants of 24-h non- or IFN-γ- and TNF-α-stimulated control or IFRD1 KD HPV16+KCs. A representative example of three different donors is shown. These data are representative for at least three independent experiments. Error bars indicate s.d. P values were determined using Welch-corrected unpaired t -tests. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Immunodetection was achieved with anti-p65 (1:1,000, sc-372, Santa Cruz), anti-phospho-p65 (Ser536; 1:1,000, #3033 Cell Signaling Technology (CST)), anti-acetyl-p65 (Lys310; 1:1,000, #3045 CST), anti-IFRD1 (1:400, T2576 Sigma-Aldrich), β-actin (1:10,000, Sigma-Aldrich) primary antibodies, and horseradish peroxidase-coupled anti-mouse (1:5,000; CST) and horseradish peroxidase-coupled anti-rabbit (1:5,000, CST) secondary antibodies.

Techniques: Microarray, Expressing, Control, Transfection, Infection, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining, Knockdown, Enzyme-linked Immunosorbent Assay, Migration

Journal: Nature Communications

Article Title: The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation

doi: 10.1038/ncomms7537

Figure Lengend Snippet: ( a ) Microarray intensities for EGFR in KCs ( n =4) and hrHPV+KCs ( n =4) represented in a box plot. ( b ) Histogram of EGFR surface protein expression on KCs and HPV16+KCs, as determined by flow cytometry. ( c ) RT–qPCR of EGFR expression in KCs transfected with complementary DNA for E2, E5, E1+E2+E6+E7 or empty control. ( d ) RT–qPCR of IFRD1 expression in KCs and HPV16+KCs treated for 72 h with 0, 0.1, 1 or 10 μg ml −1 anti-EGFR or anti-CD20. ( e ) IFRD1, RelA K310 acetylation and total RelA levels in KCs and HPV16+KCs treated for 72 h with 0, 0.1, 1 or 10 μg ml −1 anti-EGFR or anti-CD20. ( f ) Quantified protein levels of IFRD1, RelA K310 acetylation and RelA over β-actin in HPV16+KCs treated for 72 h with 0, 0.1, 1 or 10 μg ml −1 anti-EGFR (two-dimensional western blot). The expression levels of the 0 μg ml −1 -treated HPV+KCs were set as 100%. ( g ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and TNF-α-stimulated, anti-CD20- or anti-EGFR-treated HPV16+KCs (left) and KCs (right). ( h ) Enzyme-linked immunosorbent assay for CCL2, RANTES, IL-8 and CXCL9 in cleared supernatants of 24-h non- or IFN-γ- and TNF-α-stimulated, anti-CD20- or anti-EGFR-treated HPV16+KCs (left) and KCs (right). ( i ) RT–qPCR of IFRD1 expression in HPV16+KCs treated with inhibitors of PI3K (LY94002, 25 μM), mTOR (rapamycin, 50 nM), MEK1 (PD98059, 50 μM), RAF (GW5074, 20 μM) and JNK (SP60025, 20 μM). Gene expression was normalized using GAPDH as the calibrator gene. Fold changes over control were calculated and depicted. These data are representative for at least three independent experiments, except for h that was performed once. Error bars indicate s.d. P values were determined using Welch-corrected unpaired t -tests. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Immunodetection was achieved with anti-p65 (1:1,000, sc-372, Santa Cruz), anti-phospho-p65 (Ser536; 1:1,000, #3033 Cell Signaling Technology (CST)), anti-acetyl-p65 (Lys310; 1:1,000, #3045 CST), anti-IFRD1 (1:400, T2576 Sigma-Aldrich), β-actin (1:10,000, Sigma-Aldrich) primary antibodies, and horseradish peroxidase-coupled anti-mouse (1:5,000; CST) and horseradish peroxidase-coupled anti-rabbit (1:5,000, CST) secondary antibodies.

Techniques: Microarray, Expressing, Flow Cytometry, Quantitative RT-PCR, Transfection, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: Nature Communications

Article Title: The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation

doi: 10.1038/ncomms7537

Figure Lengend Snippet: ( a ) RelA K310 acetylation and total RelA levels in KCs and HPV16+KCs treated with decreasing doses of entinostat (40, 20, 10 and 2 μM), SAHA (10, 5 and 1 μM), TSA (5, 1 and 0.333 μM) or NaBu (10, 5 and 1 mM). RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in steady state ( b ) or 24-h non- or IFN-γ- and TNF-α-stimulated ( c ) control or entinostat (10 μM) pre-treated HPV16+KCs. ( d ) Total RelA levels and RelA K310 acetylation in non- or entinostat-treated control or RelA knockdown (KD) HPV16+KCs. ( e ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and TNF-α-stimulated non- or entinostat-treated control or RelA knockdown (KD) HPV16+KCs. ( f ) RT–qPCR of EGFR expression in KCs and HPV16+KCs treated with increasing doses of entinostat (0, 10 or 40 μM). Gene expression was normalized using GAPDH as the calibrator gene. ( g ) IFRD1 in control or entinostat (10 μM) pre-treated HPV16+KCs. ( h ) RT–qPCR of IFRD1 and EGFR expression in control or IFRD1 KD HPV16+KCs. Gene expression was normalized using GAPDH as the calibrator gene. Histogram ( i ) and geomean ( j ) of EGFR expression on control or IFRD1 KD HPV16+KCs, as determined by flow cytometry. s.e.m. of two independent experiments. These data are representative for at least two independent experiments. Error bars indicate s.d. P values were determined using Welch-corrected unpaired t -tests. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Immunodetection was achieved with anti-p65 (1:1,000, sc-372, Santa Cruz), anti-phospho-p65 (Ser536; 1:1,000, #3033 Cell Signaling Technology (CST)), anti-acetyl-p65 (Lys310; 1:1,000, #3045 CST), anti-IFRD1 (1:400, T2576 Sigma-Aldrich), β-actin (1:10,000, Sigma-Aldrich) primary antibodies, and horseradish peroxidase-coupled anti-mouse (1:5,000; CST) and horseradish peroxidase-coupled anti-rabbit (1:5,000, CST) secondary antibodies.

Techniques: Quantitative RT-PCR, Expressing, Control, Knockdown, Gene Expression, Flow Cytometry

Journal: Nature Communications

Article Title: The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation

doi: 10.1038/ncomms7537

Figure Lengend Snippet: ( a ) IFRD1, RelA acetylation and total RelA levels at steady state in three KC donors and three HPV16-induced CxCa lines. ( b ) RT–qPCR of IFRD1 , CCL2 , RANTES , IL-8 and CXCL9 expression, and IFRD1 protein levels in steady-state control or IFRD1 KD Caski cells. ( c ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and TNF-α-stimulated control or IFRD1 KD Caski cells. ( d ) Histogram of EGFR expression on three HPV16-induced CxCa lines. ( e ) Geomean of EGFR expression on KCs and CxCa, as determined by flow cytometry. s.e.m. of two independent experiments. ( f ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and TNF-α-stimulated anti-CD20- or anti-EGFR-treated Caski cells. ( g ) IFRD1 and RelA K310 acetylation status in Caski cells treated for 72 h with 0, 1 or 10 μg ml −1 anti-EGFR (cetuximab) or anti-CD20 (rituximab). ( h ) RT–qPCR of IFRD1 expression in KCs and Caski cells treated for 72 h with 0, 0.1, 1 or 10 μg ml −1 anti-EGFR or anti-CD20. ( i ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and TNF-α-stimulated control (dimethylsulphoxide (DMSO)) or entinostat-treated Caski cells. ( j ) Schematic representation of IFRD1-mediated RelA (de-)acetylation. (I) In KCs, RelA acetylation is positively regulated by KATs, resulting in the production of pro-inflammatory cytokines. HDACs may suppress this process. (II) In HPV+KCs, elevated EGFR levels can induce the expression of IFRD1, which can mediate RelA deacetylation by forming a bridge between RelA and HDAC1 and/or -3, hampering pro-inflammatory gene expression. (III) Interfering with EGFR signalling (1 and 2) or HDAC function (3) may lower IFRD1 levels, restoring the RelA acetylation balance, augmenting pro-inflammatory gene expression. Error bars indicate s.d. P values were determined using Welch-corrected unpaired t -tests. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Immunodetection was achieved with anti-p65 (1:1,000, sc-372, Santa Cruz), anti-phospho-p65 (Ser536; 1:1,000, #3033 Cell Signaling Technology (CST)), anti-acetyl-p65 (Lys310; 1:1,000, #3045 CST), anti-IFRD1 (1:400, T2576 Sigma-Aldrich), β-actin (1:10,000, Sigma-Aldrich) primary antibodies, and horseradish peroxidase-coupled anti-mouse (1:5,000; CST) and horseradish peroxidase-coupled anti-rabbit (1:5,000, CST) secondary antibodies.

Techniques: Quantitative RT-PCR, Expressing, Control, Flow Cytometry, Gene Expression

Journal: Cellular signalling

Article Title: The gep proto-oncogene Gα 12 mediates LPA-stimulated activation of CREB in ovarian cancer cells

doi: 10.1016/j.cellsig.2013.08.012

Figure Lengend Snippet: A. Silencing of Gα12 in HeyA8 cells using Gα12-specific shRNA was monitored by immunoblot analysis using lysates of 25 µg protein derived from three distinct clones of Gα12-silenced cells along with cells from vector control clone. B. Gα12-shRNA-HeyA8 clones were analyzed by quantitative RT-PCR for Gα12 expression. The expression levels of Gα12 for each clone in relation to vector control cells are presented in the bar graph. C. Hey cells stably expressing shRNA against Gα12 or the vector alone (non-specific scrambled shRNA vector) were serumstarved overnight. The stably silenced Gα12 cells were treated with 20 µM of LPA for 16 hours along with one group of HeyA8 cells stably-expressing the vector alone. Additionally, one group of the vector control cells was left in serum-free media for the 16-hour treatment period. After the 16-hour treatment, nuclear lysate was obtained from each cell group and analyzed by a Protein/DNA array according to manufacturer’s protocol. Representative array data from two independent experiments are presented. Each spot on the array that corresponds to a specific transcription factor was identified according to manufacturer’s protocol. Transcription factors stimulated by LPA but absent or down-regulated in Gα12-silenced cells are scored. The arrows indicate the spots corresponding to CREB. The profiles of activated transcription factors as indicated by the binding of the respective transcription factors to the DNA-elements printed in the array were analyzed in serum-starved HeyA8 cells (Upper Panel), HeyA8 cells stimulated with LPA (Middle Panel), and LPA-stimulated HeyA8 cells in which the expression of Gα12 was silenced (Lower Panel).

Article Snippet: The nuclear lysate was then analyzed for transcription factor activation using an Affymetrix Combo Protein/DNA Array (MA1215; Santa Clara, CA) according to the manufacture’s instructions.

Techniques: shRNA, Western Blot, Derivative Assay, Clone Assay, Plasmid Preparation, Quantitative RT-PCR, Expressing, Stable Transfection, DNA Array, Binding Assay

Journal: Cellular signalling

Article Title: The gep proto-oncogene Gα 12 mediates LPA-stimulated activation of CREB in ovarian cancer cells

doi: 10.1016/j.cellsig.2013.08.012

Figure Lengend Snippet: LPA-stimulated and Gα 12 -dependent Transcription Factors in HeyA8 Cells Control HeyA8 cell expressing non-specific sh-vector or HeyA8 cells in which Gα 12 were stimulated with 20 µM LPA for 16 hrs. Nuclear extracts from these cells along with unstimulated controls were analyzed for the activation of different transcription factors using “Affymetrix Combo Protein/DNA Array” as described under Methods section. Representative array data from two independent experiments are presented here. Each spot on the array, which corresponds to a specific transcription factor, was identified using the template from the user manual. The intensities of the spots were quantified using Carestream Molecular Imaging Software version 5. Transcription factors stimulated by LPA but absent or down-regulated in Gα 12 -silenced cells were scored, quantified, and tabulated.

Article Snippet: The nuclear lysate was then analyzed for transcription factor activation using an Affymetrix Combo Protein/DNA Array (MA1215; Santa Clara, CA) according to the manufacture’s instructions.

Techniques: Expressing, Activation Assay, Imaging, Software, Inhibition, Binding Assay, Methylation