Journal: PLoS ONE

Article Title: Co-Localization of the Oncogenic Transcription Factor MYCN and the DNA Methyl Binding Protein MeCP2 at Genomic Sites in Neuroblastoma

doi: 10.1371/journal.pone.0021436

Figure Lengend Snippet: (A) & (B) Three-way Venn diagrams displaying the number unique and overlapping binding sites from MYCN and MeCP2 ChIP-Chip experiments along with the hypermethylated sites identified from MeDIP analysis of the Kelly cell line, hybridized to (A) the two-array promoter set and (B) custom tiled arrays (B). (C) Pie charts representing the percentage of MYCN sites which are unique to the MYCN dataset and which overlap sites enriched for MeCP2 binding and regions of hypermethylation.

Article Snippet: Chromatin immunoprecipitations were performed using 10 μg of rabbit polyclonal anti-MeCP2 antibody (Abcam, Cambridge, UK) complexed with M-280 Sheep anti-Rabbit Dynabeads and a DynaMagTM magnetic particle concentrator (Invitrogen, Carlsbad, CA).

Techniques: Binding Assay, ChIP-chip, Methylated DNA Immunoprecipitation

Journal: PLoS ONE

Article Title: Co-Localization of the Oncogenic Transcription Factor MYCN and the DNA Methyl Binding Protein MeCP2 at Genomic Sites in Neuroblastoma

doi: 10.1371/journal.pone.0021436

Figure Lengend Snippet: (A) Array CGH analysis of the Kelly cell line showing a large 52.6MB terminal deletion on the long-arm of chromosome 18. (B) A tiled 70.5-kb section of the hemizygously deleted region on chromosome 18, the upper panels display the raw log 2 ratios and the identified, consistent binding sites for both MYCN and MeCP2. Peaks displayed are considered high confidence binding sites with an FDR score of <0.05 (red peaks) and 0.05-0.1 (orange peaks). Kelly MeDIP results are represented by the panels in blue, including the raw log 2 ratios, Kolomogorov-Smirnov test p-values (−log 10 ), and the identified peaks of hypermethylation. The lower panels show the results of a negative control experiment using normal mouse IgG. (C) A western blot of a Co-IP performed using anti-MeCP2 antibody.

Article Snippet: Chromatin immunoprecipitations were performed using 10 μg of rabbit polyclonal anti-MeCP2 antibody (Abcam, Cambridge, UK) complexed with M-280 Sheep anti-Rabbit Dynabeads and a DynaMagTM magnetic particle concentrator (Invitrogen, Carlsbad, CA).

Techniques: Binding Assay, Methylated DNA Immunoprecipitation, Negative Control, Western Blot, Co-Immunoprecipitation Assay

Journal: PLoS ONE

Article Title: Co-Localization of the Oncogenic Transcription Factor MYCN and the DNA Methyl Binding Protein MeCP2 at Genomic Sites in Neuroblastoma

doi: 10.1371/journal.pone.0021436

Figure Lengend Snippet: CpG Island Occupancy.

Article Snippet: Chromatin immunoprecipitations were performed using 10 μg of rabbit polyclonal anti-MeCP2 antibody (Abcam, Cambridge, UK) complexed with M-280 Sheep anti-Rabbit Dynabeads and a DynaMagTM magnetic particle concentrator (Invitrogen, Carlsbad, CA).

Techniques: Methylated DNA Immunoprecipitation

Journal: PLoS ONE

Article Title: Co-Localization of the Oncogenic Transcription Factor MYCN and the DNA Methyl Binding Protein MeCP2 at Genomic Sites in Neuroblastoma

doi: 10.1371/journal.pone.0021436

Figure Lengend Snippet: (A) A bar chart representing the median expression levels of genes whose promoter regions are bound by MYCN. The dataset was subdivided into unique MYCN sites, hypermethylated MYCN sites and hypermethylated MYCN sites associated with CpG Islands. (B – F) Similar analysis was performed for genes having promoters with different combinations of features (MYCN +/−; MeCP2 +/−; methylation +/− and/or CpG island +/−). Red arrows and green arrows denote the upper and lower quartiles of expression from the overall gene expression microarray results. Genes with expression values which fall in the upper quartile are considered highly expressed while genes which fall in the lower quartile are considered silent or expressed at low levels. Statistical analysis was performed using Mann-Whitney nonparametric test.

Article Snippet: Chromatin immunoprecipitations were performed using 10 μg of rabbit polyclonal anti-MeCP2 antibody (Abcam, Cambridge, UK) complexed with M-280 Sheep anti-Rabbit Dynabeads and a DynaMagTM magnetic particle concentrator (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Methylation, Gene Expression, Microarray, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Co-Localization of the Oncogenic Transcription Factor MYCN and the DNA Methyl Binding Protein MeCP2 at Genomic Sites in Neuroblastoma

doi: 10.1371/journal.pone.0021436

Figure Lengend Snippet: Comparison analysis of the top significant biological functions for (A) genes whose promoters are occupied by MYCN (B) genes whose promoters are occupied by MeCP2 (C) genes whose promoters are co-occupied by MYCN and MeCP2. Each panel represents the top five biological functions of each group of genes, and compares the significance of each of the functions across datasets. The significance scores for each biological function are represented as –log(P-value). The analysis was performed using the IPA software, significant biological functions were identified as having a p-value of less than P<0.05 (represented by a red bar).

Article Snippet: Chromatin immunoprecipitations were performed using 10 μg of rabbit polyclonal anti-MeCP2 antibody (Abcam, Cambridge, UK) complexed with M-280 Sheep anti-Rabbit Dynabeads and a DynaMagTM magnetic particle concentrator (Invitrogen, Carlsbad, CA).

Techniques: Comparison, Software

Journal: PLoS ONE

Article Title: Co-Localization of the Oncogenic Transcription Factor MYCN and the DNA Methyl Binding Protein MeCP2 at Genomic Sites in Neuroblastoma

doi: 10.1371/journal.pone.0021436

Figure Lengend Snippet: Here we illustrate the frequency, relative to background, of the various classes of canonical E-boxes (CANNTG) in non-methylated sites bound by MYCN alone (A), MeCP2 alone (C) and both MYCN and MeCP2 (B); and methylated sites bound by MYCN alone (D), MeCP2 alone (F) and both MYCN and MeCP2(E). We also include the putative MeCP2 binding motif proposed by Klose et al. (37). Motifs with 1.5 fold change over background and P<0.05 are highlighted. In non-methylated regions we see >1.5 fold-enrichment for CATCGT and CATGTG motifs. In methylated regions we see a significant preference for the CATGTG motif at locations bound by MYCN. There is a significant preference for the classical CACGTG E-box motif at methylated locations bound by MeCP2 alone.

Article Snippet: Chromatin immunoprecipitations were performed using 10 μg of rabbit polyclonal anti-MeCP2 antibody (Abcam, Cambridge, UK) complexed with M-280 Sheep anti-Rabbit Dynabeads and a DynaMagTM magnetic particle concentrator (Invitrogen, Carlsbad, CA).

Techniques: Methylation, Binding Assay

Journal: PLoS ONE

Article Title: Co-Localization of the Oncogenic Transcription Factor MYCN and the DNA Methyl Binding Protein MeCP2 at Genomic Sites in Neuroblastoma

doi: 10.1371/journal.pone.0021436

Figure Lengend Snippet: For (A) and (B), plots show increasing expression of transcription factor mRNA on the X-axis and increasing enrichment for motifs on the Y-axis. For each plot, the upper right quadrant represents greater than median expression for a transcription factor and significant over-representation of the respective binding motif (P<0.05) for (A) all commonly bound MYCN/MeCP2 sites and (B) commonly bound MYCN/MeCP2 sites only at hypermethylated promoter regions. (C) and (D) illustrate potential protein interactions for the transcription factors whose motifs were over-represented (from A and B, respectively) based on available evidence from the String database. The thickness of the edges between the nodes represents the confidence of the interaction. The MYCN/MeCP2 interaction identified in this report is shown as a dashed red line.

Article Snippet: Chromatin immunoprecipitations were performed using 10 μg of rabbit polyclonal anti-MeCP2 antibody (Abcam, Cambridge, UK) complexed with M-280 Sheep anti-Rabbit Dynabeads and a DynaMagTM magnetic particle concentrator (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Binding Assay

Journal: eLife

Article Title: Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

doi: 10.7554/eLife.03346

Figure Lengend Snippet: ( A ) Workflow for transferring mouse TF open reading frames (ORFs) into a lentiviral vector to overexpress HA-tagged TFs in 3T3-L1 cells. 750 fully sequence-verified entry TF clones were transferred using LR Gateway cloning into the Tet-On expression vector (derived from the original TRE_GOI_rtTA_hPGK vector , ‘Materials and methods’), during which the attL sites recombine with the attR sites. 734 ORF TFs were successfully transferred. ( B ) Barplots: percentage of expressed/transcribed TFs of all TFs that significantly enhance adipogenesis (positive candidates) in mouse 3T3-L1 cells based on microarray expression data in mouse 3T3-L1 (mExpr) and human hASC (hExpr) as well as POLII signal over genes (mPolII) and combined POLII signal and expression (mAny) in mouse 3T3-L1 cells ( ; ); table: positive candidates that are significantly up- or down-regulated in mouse adipose tissue compared to other probed tissues based on ArrayExpress Expression Atlas . ( C ) Protein levels of stably overexpressed HA-tagged TFs selected for follow-up in 3T3-L1 cells (follow-up TFs). The expected molecular mass for each protein is indicated above the image. Note that especially for ZEB1, several bands were detected which likely correspond to cryptic translation or specific protein degradation products given that they stem from the same open-reading frame construct and that they are all tagged by HA. ( D – F ) Relative (to control): Pparg2 ( D ), Cebpa ( E ), and Adipoq ( F ) mRNA fold-changes (FCs) in 3T3-L1 cells stably overexpressing each follow-up TF, as measured by qPCR. To measure Pparg2 mRNA levels, primers were used that target the 5′ UTR of the endogenous transcript, allowing us to differentiate between the overexpression and endogenous Pparg transcripts. ( G ) Microarray-based expression analysis of follow-up TFs during 3T3-L1 adipogenesis. In contrast to Pparg (orange), most follow-up TFs are at their maximal expression level already prior to induction of differentiation (days −2 and day 0). ( H ) Relative (to control; i.e. transduced with the shEmpty vector) Zeb1 mRNA FCs in knockdown 3T3-L1 cells at four different time points during adipogenesis, as measured by qPCR. Error bars depict the standard error of the mean from three biological replicate experiments. **p ≤ 0.01 and 0.01 < *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.03346.004

Article Snippet: A ZEB1 antibody (Santa Cruz, sc-25388, 10 μg per IP) and a rabbit isotype control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8994, 10 μg per IP) was used for each time point.

Techniques: Transferring, Plasmid Preparation, Sequencing, Clone Assay, Cloning, Expressing, Derivative Assay, Microarray, Stable Transfection, Construct, Control, Over Expression, Transduction, Knockdown

Journal: eLife

Article Title: Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

doi: 10.7554/eLife.03346

Figure Lengend Snippet: ( A ) Schematic overview of high-throughput screening illustrating how 3T3-L1 cells were transduced with 734 individual TFs in three replicates each, 3 days before induction of adipocyte differentiation (‘Materials and methods’). The effect of TF overexpression was quantified at differentiation day 7 by lipid, nucleus and cellular staining and summarized as a percentage of differentiated cells (PDC) per TF. ( B ) Overview of fold-changes (FC) compared to control for all TFs showing a differentiation FC > 1. TFs that significantly induced differentiation (FC ≥ 1.5, α = 0.05) are highlighted in red and PPARγ specifically in orange. ( C ) Effect of stably overexpressing eight putatively novel regulators of adipogenesis, PPARγ, or a control vector on 3T3-L1 differentiation as assessed by Oil Red O staining of lipid droplets at day 5 after induction. ( D ) Effect of knocking down ZEB1 or PPARγ (as a positive control), or the negative control (empty shRNA) on 3T3-L1 differentiation as assessed by Oil Red O staining at day 6 after induction. In the shRNA pool of ZEB1, shRNA2 was not used because the robustness of the cells after treatment was low. Examples of microscopic images illustrating the overexpression or knockdown (KD) effects on 3T3-L1 differentiation are shown in or , respectively. DOI: http://dx.doi.org/10.7554/eLife.03346.003

Article Snippet: A ZEB1 antibody (Santa Cruz, sc-25388, 10 μg per IP) and a rabbit isotype control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8994, 10 μg per IP) was used for each time point.

Techniques: High Throughput Screening Assay, Transduction, Over Expression, Staining, Control, Stable Transfection, Plasmid Preparation, Positive Control, Negative Control, shRNA, Knockdown

Journal: eLife

Article Title: Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

doi: 10.7554/eLife.03346

Figure Lengend Snippet: ( A ) Relative (to day −2) Zeb1 mRNA levels in wild-type 3T3-L1 cells during differentiation, as measured by qPCR. ( B ) Raw Ct values for Pparg , Zeb1 as well as the housekeeping gene HPRT1 at days 0 and 4 of 3T3-L1 differentiation as measured by qPCR. ( C ) Pparg and Zeb1 mRNA levels in pre-adipocytes and adipocytes derived from publicly available data through ArrayExpress . ( D ) Protein levels (fmol/μg nuclear extract) of ZEB1 during 3T3-L1 differentiation (biological replicate of data shown in ). ( E ) Pparg2 and Cebpa mRNA levels after ZEB1 knockdown and overexpression at day 4 after adipogenic induction as measured by qPCR. ( F ) Fold-changes of expression levels of selected adipogenic factors in response to ZEB1 KD as measured by qPCR and RNA-seq at day 0 and day 2 of 3T3-L1 differentiation. The Pearson's correlation coefficient (r) is indicated. ( G ) Number of significantly up- and down-regulated genes after ZEB1 knockdown belonging to previously defined expression clusters (2/5/Low/1) . The typical expression pattern of genes in each cluster is sketched. Clusters are sorted by decreasing enrichment of up-regulated genes and corresponding p-values (chi-square test) are listed. ( H ) Changes in mRNA levels and POLII binding over gene bodies after ZEB1 and SMRT knockdown, respectively . The Spearman's ρ is indicated, showing a significantly negative correlation. ( I ) Percent of genes with dynamic SMRT binding during adipogenesis (red), of genes that lose/gain POLII upon SMRT KD (red), and of random genes (grey) that are significantly differentially expressed upon ZEB1 KD. Error bars depict the standard error of the mean. **p ≤ 0.01 and 0.01< *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.03346.008

Article Snippet: A ZEB1 antibody (Santa Cruz, sc-25388, 10 μg per IP) and a rabbit isotype control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8994, 10 μg per IP) was used for each time point.

Techniques: Derivative Assay, Knockdown, Over Expression, Expressing, RNA Sequencing, Binding Assay

Journal: eLife

Article Title: Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

doi: 10.7554/eLife.03346

Figure Lengend Snippet: ( A ) Protein levels (fmol/μg nuclear extract) of ZEB1 during 3T3-L1 differentiation (one representative biological replicate). ( B ) Pparg2 and Cebpa mRNA levels after ZEB1 knockdown and overexpression in un-induced 3T3-L1 pre-adipocytes as measured by qPCR. ( C ) Expression levels [ln(FPKM), ‘Materials and methods’] of mouse genes in ZEB1 KD vs. control cells at day 0 and day 2 after differentiation induction as measured by RNA-seq. Significantly up-regulated genes (FC ≥ 1.5, padj ≤ 0.01) are highlighted in blue, down-regulated genes in orange (FC ≤ 0.67, padj ≤ 0.01), significantly de-regulated follow-up TFs as well as adipogenic TFs such as PPARγ and C/EBPs are indicated in black. Bar plots represent the percentage of genes that are significantly up- or down-regulated. Representative enriched GeneGO pathway categories for up- or down-regulated genes are highlighted (complete results in ). ( D ) Number of significantly up- or down-regulated genes belonging to previously defined expression clusters (High/7/4/3) . The typical expression pattern of genes in each cluster as well as of representative members that are significantly down-regulated upon ZEB1 KD is sketched. Clusters are sorted by increasing enrichment of down-regulated genes and corresponding p-values (chi-square test) are listed. ( E ) Distribution of gene expression FCs at day 0 after ZEB1 KD for genes annotated as positive or negative regulators of adipogenesis . Error bars depict the standard error of the mean. **p ≤ 0.01 and 0.01 < *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.03346.007

Article Snippet: A ZEB1 antibody (Santa Cruz, sc-25388, 10 μg per IP) and a rabbit isotype control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8994, 10 μg per IP) was used for each time point.

Techniques: Knockdown, Over Expression, Expressing, Control, RNA Sequencing, Gene Expression

Journal: eLife

Article Title: Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

doi: 10.7554/eLife.03346

Figure Lengend Snippet: ( A ) ZEB1, C/EBPβ, POLII, and Control (CTRL) read density tracks at the Pparg locus. ( B ) Number of ZEB1-bound regions and of their proximal (≤ 10 kb) genes in 3T3-L1 cells. Distribution of ZEB1 binding with respect to genomic annotation (‘Materials and methods’). ( C ) De novo motif discovery using MEME and a 50 bp sequence centered on ZEB1 peak summits reveals the canonical ZEB1 motif (p = 10 −8 , ‘Materials and methods’) ( D ) Motif enrichment analysis in a 100 bp window around ZEB1 peak summits reveals 138 significantly enriched motifs (complete results in ). Highlighted here are motif names of the known early adipogenic regulators C/EBPβ, NFI, and AP1 factors as well as RUNX and SMAD3. ( E ) Peak overlap between ZEB1 and C/EBPβ (day 0) as well as AP1 factors (day 0, 4 hr) in 3T3-L1 cells. ( F ) Overview of ZEB1, C/EBPβ, AP1 proteins ATF2 and ATF7, POLII normalized ChIP-seq as well as DNase-seq (DHS) enrichments (‘Materials and methods’) in a 2 kb window around the summits of ZEB1 peaks that overlap C/EBPβ binding. Intervals are sorted based on decreasing ZEB1 enrichment. ( G ) Summarized results from mass spectrometry experiments of proteins that were identified when pulling down ZEB1 (complete results in ). DOI: http://dx.doi.org/10.7554/eLife.03346.009

Article Snippet: A ZEB1 antibody (Santa Cruz, sc-25388, 10 μg per IP) and a rabbit isotype control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8994, 10 μg per IP) was used for each time point.

Techniques: Control, Binding Assay, Sequencing, ChIP-sequencing, Mass Spectrometry

Journal: eLife

Article Title: Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

doi: 10.7554/eLife.03346

Figure Lengend Snippet: ( A ) ZEB1 ChIP-qPCR validation of ChIP-seq data at 12 selected ZEB1 target sites and three negative control (CTRL) regions during 3T3-L1 adipogenesis. ( B ) Scatterplot and Spearman's ρ of ZEB1 ChIP-seq and ZEB1-HA ChIP-seq read counts inside genomic intervals defined by ZEB1 binding in pre-adipocytes. ( C ) Spearman correlations between read counts for replicate ZEB1 ChIP-seq (including ZEB1-HA) as well as publicly available POLII and DNase-seq data ( ; ) inside genomic intervals defined by ZEB1 binding in pre-adipocytes. ( D ) Distribution of randomly shifted ZEB1, C/EBPβ, and POLII peaks with respect to genomic annotation (‘Materials and methods’). ( E ) ZEB1 motif density at 800 bp centered on ZEB1 peak summits. ( F ) Fraction of ZEB1 and randomly shifted ZEB1 peaks (to show background values) that contain at least one or two, respectively, ZEB1, CACCTG (E-box), C/EBPβ, AP1, NFIC and SMAD3 motif hits (‘Materials and methods’). ( G ) Peak overlap between randomly shifted ZEB1 and C/EBPβ bound regions in 3T3-L1 pre-adipocytes. ( H ) Overview of ZEB1, ZEB1-HA, C/EBPβ, AP1 factors ATF2 and ATF7, POLII and H3K9AC normalized ChIP-seq as well as DNase-seq and control (CTRL) enrichments (‘Materials and methods’) in a 2 kb window around the summits of ZEB1 peaks. Intervals are sorted based on decreasing ZEB1 enrichment. ( I ) Mean C/EBPβ and AP1 complex proteins JUN and FOSL normalized (to total read number) ChIP-seq enrichments in human HepG2 and lymphoblastoid cell lines (LCLs) in a 8 kb window around the summits of ZEB1 peaks detected in LCLs. DOI: http://dx.doi.org/10.7554/eLife.03346.010

Article Snippet: A ZEB1 antibody (Santa Cruz, sc-25388, 10 μg per IP) and a rabbit isotype control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8994, 10 μg per IP) was used for each time point.

Techniques: ChIP-qPCR, Biomarker Discovery, ChIP-sequencing, Negative Control, Binding Assay, Control

Journal: eLife

Article Title: Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

doi: 10.7554/eLife.03346

Figure Lengend Snippet: ( A ) C/EBPβ expression (mRNA level) in stable C/EBPβ KD and control 3T3-L1 pre-adipocytes as measured by qPCR. ( B ) C/EBPβ ChIP-qPCR at 10 C/EBPβ-ZEB1, 6 ZEB1-only and six negative control regions according to our ZEB1 D0 ChIP-seq data and publicly available C/EBPβ ChIP-seq data . 5 out of 6 ZEB1-only regions also show C/EBPβ ChIP enrichment. *C/EBPβ-enriched regions ( C ) ZEB1 ChIP-qPCR at 9 C/EBPβ-ZEB1 regions as well as one ZEB1-only region (10) in C/EBPβ KD and control 3T3-L1 cells. * regions showing changes in ZEB1 enrichment after C/EBPβ KD ( D ) Zeb1 expression (mRNA level) in stable C/EBPβ KD and control 3T3-L1 cells as measured by qPCR. ( E ) POLII, ZEB1, and C/EBPβ read density tracks at the Zeb1 locus in 3T3-L1 pre-adipocytes. DOI: http://dx.doi.org/10.7554/eLife.03346.011

Article Snippet: A ZEB1 antibody (Santa Cruz, sc-25388, 10 μg per IP) and a rabbit isotype control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8994, 10 μg per IP) was used for each time point.

Techniques: Expressing, Control, ChIP-qPCR, Negative Control, ChIP-sequencing

Journal: eLife

Article Title: Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

doi: 10.7554/eLife.03346

Figure Lengend Snippet: ( A ) Overview of ZEB1, C/EBPβ, RXRα, PPARγ, POLII normalized ChIP-seq as well as DNase-seq enrichments (‘Materials and methods’) in a 2 kb window around the summits of static ( padj ≥ 0.1 or FC < 2) and early-only (days-2 and 0 but not days 2 and 4; padj ≤ 0.1, FC ≥ 2) ZEB1-bound regions during 3T3-L1 differentiation. ( B ) Spearman correlations between read counts for ZEB1 ChIP-seq data at distinct adipogenic time points (days −2, 0, 2, and 4) inside genomic intervals defined by ZEB1 binding at any of these time points. ( C ) ZEB1 and POLII read density tracks at the Zbtb16 and Pparg loci during 3T3-L1 differentiation (days-2, 0, 2, and 4). Summarized genome-wide results are included in . ( D ) Differential motif discovery using MEME and a 50 bp sequence centered on summits of early-only vs static ZEB1 peaks reveals non-adipogenic motifs: RUNX1/2 and TEAD1 (p < 10 −5 , ‘Materials and methods’). ( E ) GREAT-based Gene Ontology enrichment analysis of genes associated with early-only vs static ZEB1 binding reveals terms associated with chemokine secretion and non-adipogenic functions. Full results are displayed in . ( F ) Number of significantly up- or down-regulated genes associated (≤10 kb) with at least one early-only or late-only ZEB1 bound region, respectively, belonging to previously defined expression clusters (1/Low/7/5) . The typical expression pattern of genes in each cluster is sketched. Clusters are sorted by increasing enrichment of late-only ZEB1-bound genes and corresponding p-values (chi-square test) are listed. Only clusters showing a highly significant p-value (p < 10 −10 ) are shown. ( G ) Fraction of genes associated with early-only, late-only, and static ZEB1 binding as well as the fraction of all genes significantly up (blue) and down (orange)-regulated after ZEB1 KD as measured at differentiation days 0 and 2. DOI: http://dx.doi.org/10.7554/eLife.03346.013

Article Snippet: A ZEB1 antibody (Santa Cruz, sc-25388, 10 μg per IP) and a rabbit isotype control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8994, 10 μg per IP) was used for each time point.

Techniques: ChIP-sequencing, Binding Assay, Genome Wide, Sequencing, Expressing

Journal: eLife

Article Title: Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

doi: 10.7554/eLife.03346

Figure Lengend Snippet: ( A ) ZEB1 and POLII read density tracks at the Klf15 locus during 3T3-L1 differentiation (days −2, 0, 2, and 4). Late-only bound regions are highlighted. ( B ) ZEB1, C/EBPβ, RXRα, PPARγ, POLII normalized ChIP (‘Materials and methods’) as well as DHS enrichments in a 2 kb window around the summits of late-only (days 2 and 4 but not days −2 and 0; padj ≤ 0.1, FC ≥ 2) ZEB1-bound regions during 3T3-L1 differentiation. ( C ) Differential motif discovery using MEME and a 50 bp sequence centered on summits of late-only vs. static ZEB1 peaks reveals adipogenic motifs: C/EBPα|C/EBPβ, NFIC and PPARG::RXR (p < 10 −3 , ‘Materials and methods’). ( D ) GREAT-based Gene Ontology enrichment analysis of genes associated with late-only vs. static ZEB1 binding reveals terms associated with fat cell differentiation and function (complete results in ). ( E ) Fraction of genes associated with late-only ZEB1 binding and fraction of all genes significantly up (blue) and down (orange)-regulated after ZEB1 KD as measured at differentiation day 2 (complete results in ). DOI: http://dx.doi.org/10.7554/eLife.03346.012

Article Snippet: A ZEB1 antibody (Santa Cruz, sc-25388, 10 μg per IP) and a rabbit isotype control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8994, 10 μg per IP) was used for each time point.

Techniques: Sequencing, Binding Assay, Cell Differentiation

Journal: eLife

Article Title: Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

doi: 10.7554/eLife.03346

Figure Lengend Snippet: ( A ) Effect of ZEB1 knockdown on the adipogenic gene regulatory network. The network was assembled on the ‘Adipogenesis’ Pathway scaffold in WikiPathways as well as reviews and most recent publications of novel adipogenic regulators ( ; ; ). ZEB1 and C/EBPβ-bound regions that are proximal (within 500 bp) to TSSs or genes in pre-adipocytes are highlighted. *Significant ( padj ≤ 0.01) expression changes after ZEB1 KD at day 0 of 3T3-L1 differentiation. Other candidate adipogenic regulators identified by our high throughput screen are listed. ( B ) Expression changes of adipogenic commitment genes after ZEB1 KD in 3T3-L1 pre-adipocytes as measured by RNA-seq. Displayed genes are either part of the pre-adipocyte expression signature derived by or of the list of pre-adipocyte commitment factors compiled by . Lpl and Igfbp4 occur in both lists. Significant differences in expression ( padj ≤ 0.01) are marked in orange (FC ≤ 0.67) and blue (FC ≥ 1.5). Black-grey squares depict ZEB1 binding to TSSs or gene bodies. ( C ) Effect of ZEB1 knockdown and overexpression on C3H10T1/2 adipogenesis as assessed by Oil Red O staining at day 7 and day 8, respectively after induction. DOI: http://dx.doi.org/10.7554/eLife.03346.014

Article Snippet: A ZEB1 antibody (Santa Cruz, sc-25388, 10 μg per IP) and a rabbit isotype control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8994, 10 μg per IP) was used for each time point.

Techniques: Knockdown, Expressing, High Throughput Screening Assay, RNA Sequencing, Derivative Assay, Binding Assay, Over Expression, Staining

Journal: eLife

Article Title: Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

doi: 10.7554/eLife.03346

Figure Lengend Snippet: ( A ) Effect of ZEB1 knockdown on the expression [log2 (FC) mRNA] of adipogenic ( Adipoq, Cebpa, Ebf1, Pparg2 ) and EMT ( Snai1, Snai2, Twist1 ) factors as measured by qPCR at day 8 after induction. ( B ) Effect of ZEB1 overexpression on the expression [log2 (FC) mRNA] of adipogenic ( Cebpa, Ebf1, Pparg2 ), pre-adipogenic ( Zfp423, Zfp521 ), and EMT ( Snai1, Twist1 ) factors as measured by qPCR at day 0 after induction of differentiation. ( C ) Western Blot showing PPARγ induction upon ZEB1 overexpression (visualized using anti-HA antibody) in C3H10T1/2 cells using PCNA as a normalization control. R1-3 indicates biological replicates. Error bars depict the standard error of the mean. **p ≤ 0.01 and 0.01 < *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.03346.015

Article Snippet: A ZEB1 antibody (Santa Cruz, sc-25388, 10 μg per IP) and a rabbit isotype control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8994, 10 μg per IP) was used for each time point.

Techniques: Knockdown, Expressing, Over Expression, Western Blot, Control

Journal: eLife

Article Title: Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

doi: 10.7554/eLife.03346

Figure Lengend Snippet: ( A and B ) Adipocyte differentiation in stromal vascular fraction (SVF) transplants from different donor mice (as indicated) fed a high-fat diet for 6 weeks . ( A ) Fat sections from representative samples of ZEB1-overexpressing and control SVF transplants stained with Hematoxylin (blue) and Eosin (pink). ( B ) Fat cell content of the transplanted SVF cells containing ZEB1 and control overexpression or knockdown constructs. Error bars depict the standard error of the mean. *p = 0.05, one-sided Wilcoxon-rank sum test. ( C ) Zeb1 mRNA expression normalized to 36B4 in human subcutaneous SVF of obese subjects plotted against percent ex vivo differentiated adipocytes of human subcutaneous SVF, subject fat mass, and adiponectin levels. Spearman's ρ is indicated, **p ≤ 0.01 and 0.01 < *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.03346.016

Article Snippet: A ZEB1 antibody (Santa Cruz, sc-25388, 10 μg per IP) and a rabbit isotype control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8994, 10 μg per IP) was used for each time point.

Techniques: Control, Staining, Over Expression, Knockdown, Construct, Expressing, Ex Vivo

Journal: eLife

Article Title: Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

doi: 10.7554/eLife.03346

Figure Lengend Snippet: ( A ) Hematoxylin (blue) and Eosin (pink)-stained fat sections from representative samples of mouse ZEB1 knockdown transplants as well as the corresponding control (scrambled siRNA). ( B ) Number of nuclei per section (average over three sections) from ZEB1 overexpression, KD, or control SVF transplants. DOI: http://dx.doi.org/10.7554/eLife.03346.017

Article Snippet: A ZEB1 antibody (Santa Cruz, sc-25388, 10 μg per IP) and a rabbit isotype control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8994, 10 μg per IP) was used for each time point.

Techniques: Staining, Knockdown, Control, Over Expression

Journal: Cancer cell

Article Title: MUTANT EZH2 INDUCES A PRE-MALIGNANT LYMPHOMA NICHE BY REPROGRAMMING THE IMMUNE RESPONSE

doi: 10.1016/j.ccell.2020.04.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit H3K27me3 (for ChIP) , Cell Signaling , Cat# 9733.

Techniques: Control, Blocking Assay, Recombinant, Adjuvant, Plasmid Preparation, Binding Assay, Staining, RNA Library Preparation, MicroChIP Assay, Sequencing, Microarray, Knock-In, Software, Gene Expression, Targeted Proteomics

Journal: Oncology Letters

Article Title: lncRNA-CD160 decreases the immunity of CD8 + T cells through epigenetic mechanisms in hepatitis B virus infection

doi: 10.3892/ol.2020.11534

Figure Lengend Snippet: Percentage of CD160 + CD8 + T cells in patients with CHB with different natural history is negatively associated with the progress of CHB. (A) The percentage of CD160 + CD8 + T cells in patients with CHB was detected using a FACSCalibur flow cytometer, and statistical analysis was performed. (B) The percentage of CD160 + CD8 + T cells in patients with different stages of CHB was detected. (C) Analysis of the percentage of CD160 + CD8 + T cells in patients with different stages of CHB. (D) The expression of CD160 was inhibited by CD160-siRNA. (E) CD160 + CD8 + T cells were transfected with CD160-siRNA, and the CD160-siRNA significantly inhibited the expression of CD160. Following inhibition of CD160, (F) the expression of SAP was reduced and (G) the percentage of SAP + CD160 + cells in total CD8 + T cells was inhibited. To further clarify the role of CD160 in the CD8 + T cell immune response, the concentrations of (H) IFN-γ and (I) TNF-α were detected, which are produced by CD8 + T cells. IFN-γ and TNF-α were significantly decreased following CD160-knockdown in CD8 + T cells. **P<0.01, ***P<0.005. HBV, hepatitis B virus; CHB, chronic HBV; siRNA, small interfering RNA; SAP, (SLAM)-associated protein con, control; IT, immune tolerance; LR, low-replicate; IC, immunological clearance.

Article Snippet: The sections were then incubated at 4°C overnight with CD160 monoclonal mouse anti-human IgG (1:100; cat. no. AF3899; R&D Systems, Inc.) in a 1:50 dilution with 5% skimmed milk PBS buffer, followed by incubation with the corresponding secondary goat anti-mouse IgG-HRP antibody at room temperature (1:300; sc-2005; Santa Cruz Biotechnology, Inc.) for 45 min.

Techniques: Flow Cytometry, Expressing, Transfection, Inhibition, Produced, Knockdown, Virus, Small Interfering RNA, Control

Journal: Oncology Letters

Article Title: lncRNA-CD160 decreases the immunity of CD8 + T cells through epigenetic mechanisms in hepatitis B virus infection

doi: 10.3892/ol.2020.11534

Figure Lengend Snippet: CD160 inhibits HDAC11 expression via epigenetic regulation in CD8 + T cells. (A) A gene microarray assay was conducted with CD160 + CD8 + T cells and CD160 − CD8 + T cells, which were isolated from patients with chronic hepatitis B virus, with unsupervised clustering analysis. Green indicates decreased expression and red indicates increased expression. (B) Gene microarray assay with supervised clustering analysis was performed with CD160 + CD8 + T cells and CD160 − CD8 + T cells for epigenetic factor detection. (C) The expression of HDAC11 in CD160 + CD8 + T cells and CD160 − CD8 + T cells was detected by RT-qPCR assay. (D) An immunofluorescence assay for CD8, CD160 and HDAC11 detection was performed with CD160 + CD8 + T cells to obtain confocal microscopic images; magnification, ×1,000. (E) CD8 + T cells were transfected with CD160-siRNA and the expression of HDAC11 was detected by RT-qPCR, and the expression level of HDAC11 was negatively associated with the expression of CD160. (F) The protein level of HDAC11 was measured by western blotting following transfection of CD8 + T cells with CD160-siRNA. (G) The expression of HDAC11 following transfection with HDAC11 siRNA. CD8 + T cells were transfected with HDAC11-siRNA and the expression levels of (H) IFN-γ and (I) TNF-α were detected by RT-qPCR assay. (J) Flow cytometry was performed to For detect the percentage of HDAC11 +/− CD160 +/− CD8 + T cells in the total CD8 + T cell population. *P<0.05, ***P<0.005. HDAC11, histone-modification enzyme gene histone deacetylases 11; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA; con, control.

Article Snippet: The sections were then incubated at 4°C overnight with CD160 monoclonal mouse anti-human IgG (1:100; cat. no. AF3899; R&D Systems, Inc.) in a 1:50 dilution with 5% skimmed milk PBS buffer, followed by incubation with the corresponding secondary goat anti-mouse IgG-HRP antibody at room temperature (1:300; sc-2005; Santa Cruz Biotechnology, Inc.) for 45 min.

Techniques: Expressing, Microarray, Isolation, Virus, Quantitative RT-PCR, Immunofluorescence, Transfection, Western Blot, Flow Cytometry, Modification, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA, Control

Journal: Oncology Letters

Article Title: lncRNA-CD160 decreases the immunity of CD8 + T cells through epigenetic mechanisms in hepatitis B virus infection

doi: 10.3892/ol.2020.11534

Figure Lengend Snippet: lncRNA-CD160 expression is positively associated with CD160 expression in CD8 + T cells. (A) lncRNA gene microarray assay was conducted with CD160 + CD8 + T cells and CD160 − CD8 + T cells, which were isolated from patients with chronic HBV, with unsupervised clustering analysis. Green indicates decreased expression and red indicates increased expression. (B) lncRNA gene microarray assay with supervised clustering analysis was performed with CD160 + CD8 + T cells and CD160 − CD8 + T cells. (C) Reverse transcription-qPCR assay was performed to detect the lncRNA-CD160 expression level in CD160 +/− CD8 + T cells. (D) Chromosome analysis indicated that both CD160 and lncRNA-CD160 were located at Chr1q42.3, and lncRNA-CD160 was partly located at the region of CD160, which was between the B and C region; therefore, lncRNA-CD160 could also be termed lncRNA-CD160. Chromatin immunoprecipitation-qPCR was performed to investigate the relationship between (E) lncRNA-CD160 and H3K9Me1, (F) the relationship between lncRNA-CD160 and HDAC11 also was detected. HDAC11 and H3K9Me1 trimethylation levels were promoted in the lncRNA-CD160 loci. *P<0.05, **P<0.01, ***P<0.005. qPCR, quantitative PCR; lncRNA, long non-coding RNA; HBV, hepatitis B virus; HDAC11, histone-modification enzyme gene histone deacetylases 11.

Article Snippet: The sections were then incubated at 4°C overnight with CD160 monoclonal mouse anti-human IgG (1:100; cat. no. AF3899; R&D Systems, Inc.) in a 1:50 dilution with 5% skimmed milk PBS buffer, followed by incubation with the corresponding secondary goat anti-mouse IgG-HRP antibody at room temperature (1:300; sc-2005; Santa Cruz Biotechnology, Inc.) for 45 min.

Techniques: Expressing, Microarray, Isolation, Reverse Transcription, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Virus, Modification

Journal: Oncology Letters

Article Title: lncRNA-CD160 decreases the immunity of CD8 + T cells through epigenetic mechanisms in hepatitis B virus infection

doi: 10.3892/ol.2020.11534

Figure Lengend Snippet: lncRNA-CD160 inhibits IFN-γ and TNF-α secretion in CD8 + T cells via epigenetic regulation. In order to demonstrate the role of lncRNA-CD160 on IFN-γ and TNF-α secretion, siRNA targeting lncRNA-CD160 was transfected into the CD8 + T cells. (A) The efficiency of lncRNA-CD160 siRNA was detected, and the concentrations of (B) IFN-γ and (C) TNF-α were detected by ELISA assay. A CHIP-qPCR assay was performed to demonstrate the mechanism of the IFN-γ and TNF-α secretion inhibition. When lncRNA-CD160 was knocked down, the H3K9Me1 expression levels, which could be mediated by HDAC11 at the (D) IFN-γ and (E) TNF-α promoters loci, were significant inhibited. (F) Immunoprecipitation and western blot assays were performed to detect the expression of HDAC11 in the immunoprecipitate using an anti-HDAC11-specific antibody. (G) Gel electrophoresis and (H) an image of biotinylated lncRNA-CD160. (I) Reverse transcription-qPCR analysis of lncRNA-CD160 retrieved by IgG or anti-HDAC11 from CD8 + T-cell lysates of patients with HBV. (J) FISH following lncRNA-CD160 siRNA transfection, magnification, ×1,000. (K) RNA pull-down and western blot assays were conducted to investigate the association between lncRNA-CD160 and HDAC11, and the data indicated that lncRNA-CD160 and HDAC11 could bind to each other. (L) Further RNA FISH and immunofluorescence analyses were performed to investigate the locations of lncRNA-CD160 and HDAC11, and the results demonstrated that both were located in the nucleus of CD8 + T cells, magnification, ×1,000. A CHIP-qPCR assay was also performed to reveal the location of the lncRNA-CD160 and HDAC11 complex, and the results revealed that lncRNA-CD160-siRNA could significantly inhibit the expression of HDAC11 at (M) IFN-γ and (N) TNF-α promoter regions. **P<0.01, ***P<0.005. FISH, fluorescent in situ hybridization; lncRNA, long non-coding RNA; con, control; siRNA, small interfering RNA; qPCR, quantitative PCR; HDAC11, histone-modification enzyme gene histone deacetylases 11.

Article Snippet: The sections were then incubated at 4°C overnight with CD160 monoclonal mouse anti-human IgG (1:100; cat. no. AF3899; R&D Systems, Inc.) in a 1:50 dilution with 5% skimmed milk PBS buffer, followed by incubation with the corresponding secondary goat anti-mouse IgG-HRP antibody at room temperature (1:300; sc-2005; Santa Cruz Biotechnology, Inc.) for 45 min.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, ChIP-qPCR, Inhibition, Expressing, Immunoprecipitation, Western Blot, Nucleic Acid Electrophoresis, Reverse Transcription, Immunofluorescence, In Situ Hybridization, Control, Small Interfering RNA, Real-time Polymerase Chain Reaction, Modification

Journal: Oncology Letters

Article Title: lncRNA-CD160 decreases the immunity of CD8 + T cells through epigenetic mechanisms in hepatitis B virus infection

doi: 10.3892/ol.2020.11534

Figure Lengend Snippet: lncRNA-CD160 suppresses HBV replication during infection in vivo . (A) To investigate the effect of lncRNA-CD160 on HBV replication, an adoptive transfer model was established. (B) Following adoptive transfer, the serum HBsAg levels were detected at different time points using a Roche Cobas 6000 immuno-chemiluminescence analyzer. *P<0.05, ***P<0.005 vs. LV-lncRNA-CD160. (C) The HBV DNA load was detected by reverse transcription--quantitative PCR assay at different time points following adoptive transfer. *P<0.05, **P<0.01 vs. LV-lncRNA-CD160. (D) An immunohistochemistry assay was performed for HBcAg detection in the liver tissues, which were harvested from the adoptive transfer model mice, magnification, ×1,000. (E) The percentages of HBcAg-positive hepatocytes were quantified. **P<0.01 and ***P<0.005. (F) An overview of the role of lncRNA-CD160 in the mediation of IFN-γ and TNF-α. lncRNA, long non-coding RNA; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; LV, lentivirus; HBcAg, hepatitis B virus c antibody; SAP, (SLAM)-associated protein; siRNA, small interfering RNA.

Article Snippet: The sections were then incubated at 4°C overnight with CD160 monoclonal mouse anti-human IgG (1:100; cat. no. AF3899; R&D Systems, Inc.) in a 1:50 dilution with 5% skimmed milk PBS buffer, followed by incubation with the corresponding secondary goat anti-mouse IgG-HRP antibody at room temperature (1:300; sc-2005; Santa Cruz Biotechnology, Inc.) for 45 min.

Techniques: Infection, In Vivo, Adoptive Transfer Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunohistochemistry, Virus, Small Interfering RNA

Journal: Cancer cell

Article Title: Binary pan-cancer classes with distinct vulnerabilities defined by pro- or anti-cancer YAP/TEAD activity

doi: 10.1016/j.ccell.2021.06.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ChIP, MeDIP and ChIP-seq For TEAD4 ChIPs, control or YAP-expressing SCLC and retinoblastoma lines were harvested 5 days after viral transduction and processed for ChIP ( Ni and Bremner, 2007 ; Ni et al., 2008 ) with 2 µg of antibody and 20 µl of Dynabeads (sheep anti-mouse or anti-rabbit, Invitrogen).

Techniques: Plasmid Preparation, Derivative Assay, Recombinant, Electron Microscopy, Staining, SYBR Green Assay, Sample Prep, Microarray, Expressing, CRISPR, Software, Flow Cytometry

Journal: Cell Cycle

Article Title: Repression by RB1 characterizes genes involved in the penultimate stage of erythroid development

doi: 10.1080/15384101.2015.1090067

Figure Lengend Snippet: Midgestational Rb1 family erythroid TKO embryos are viable

Article Snippet: We used the following primary antibodies: RB1 (554136), Ter119 (553670), N-terminal p130 (610262) from BD Transduction Labs; p107 and C-terminal p130 (C-20) from Santa Cruz; β-actin (A5441) from Sigma.

Techniques:

Journal: Cell Cycle

Article Title: Repression by RB1 characterizes genes involved in the penultimate stage of erythroid development

doi: 10.1080/15384101.2015.1090067

Figure Lengend Snippet: Genome-wide effects of RB1 deficiency. (A) Butterfly plots comparing microarray results from pocket protein deficient FLC to controls employing the Signal-to-noise (S2N) metric. The blue line corresponds to downregulated genes (the 300 most negative S2N values), the red line to upregulated genes (the 300 most positive S2N values), in rank order. The green, tan, and black lines represent 50%, 5%, and 1% thresholds, respectively, generated by permutation of randomized columnar data. The barbed arrow points a shoulder of several hundred genes expressed above the 1% threshold in the absence of RB1. A second arrow points to the position of the gene most repressed in the absence of p107, Serinc3. (B) Butterfly plots comparing microarray results from pocket protein deficient FLC to controls, in culture for 0, 24, and 48 hours. All time points were combined in calculating the S2N metric. The labels and scale are the same as in panel A.

Article Snippet: We used the following primary antibodies: RB1 (554136), Ter119 (553670), N-terminal p130 (610262) from BD Transduction Labs; p107 and C-terminal p130 (C-20) from Santa Cruz; β-actin (A5441) from Sigma.

Techniques: Genome Wide, Microarray, Generated

Journal: Cell Cycle

Article Title: Repression by RB1 characterizes genes involved in the penultimate stage of erythroid development

doi: 10.1080/15384101.2015.1090067

Figure Lengend Snippet: Clustering analysis. (Top) Heat map of microarray results comparing RB1-deficient FLC in culture to controls. The results show the expression patterns of genes in control cells cultured for 0 hours (5 lanes), 24 hours (4 lanes), and 48 hours (3 lanes). Z-scores are gene normalized over the time course, using only the control samples. The data is divided into 16 bins. The genes are binned according to their regulation by RB1, employing the S2N metric (combining all time points), with those most derepressed in the absence of RB1 in bin 1 and those most repressed in bin 16. There are 1,000 unique genes per bin, except for bin 16, which has 1,047 genes. See supplemental file S1 for greater detail. (Bottom) Color coding and labeling of the major expression pattern clusters.

Article Snippet: We used the following primary antibodies: RB1 (554136), Ter119 (553670), N-terminal p130 (610262) from BD Transduction Labs; p107 and C-terminal p130 (C-20) from Santa Cruz; β-actin (A5441) from Sigma.

Techniques: Microarray, Expressing, Cell Culture, Labeling

Journal: Cell Cycle

Article Title: Repression by RB1 characterizes genes involved in the penultimate stage of erythroid development

doi: 10.1080/15384101.2015.1090067

Figure Lengend Snippet: RB1-repressed genes are well expressed but downregulated late in development. (A) Charts show the mean Z-scores of genes in each of the major clusters in the control samples at 0 hours, 24 hours, and 48 hours. The chart at the lower right shows the Z-scores of the genes in bin 1 (clusters A, C, and D), in the control and RB1 deleted (KO) samples, at the same time points. Beneath the charts, the table shows the number of genes and relative median expression of all the genes in each cluster. (B) Bar graphs show the number of genes in each bin and cluster that satisfy the indicated condition. Present and absent calls are determined by Affymetrix software. The color schemes are consistent throughout. The bar graph at the lower right shows the percentage of genes in each cluster with 3 absent calls, at each time point.

Article Snippet: We used the following primary antibodies: RB1 (554136), Ter119 (553670), N-terminal p130 (610262) from BD Transduction Labs; p107 and C-terminal p130 (C-20) from Santa Cruz; β-actin (A5441) from Sigma.

Techniques: Expressing, Software

Journal: Cell Cycle

Article Title: Repression by RB1 characterizes genes involved in the penultimate stage of erythroid development

doi: 10.1080/15384101.2015.1090067

Figure Lengend Snippet: Repression by RB1 correlates with E2F binding. Comparison to external ChIP-chip, ChIP-Seq, and alternative splicing data sets. The bar graphs show the number of genes in each bin and cluster bound by the protein indicated at the top. H3K27me3 (down) represents genes exhibiting downregulation of this histone mark at the Ter119- to Ter119+ transition. Color schemes are consistent with previous figures.

Article Snippet: We used the following primary antibodies: RB1 (554136), Ter119 (553670), N-terminal p130 (610262) from BD Transduction Labs; p107 and C-terminal p130 (C-20) from Santa Cruz; β-actin (A5441) from Sigma.

Techniques: Binding Assay, ChIP-chip, ChIP-sequencing

Journal: Cell Cycle

Article Title: Repression by RB1 characterizes genes involved in the penultimate stage of erythroid development

doi: 10.1080/15384101.2015.1090067

Figure Lengend Snippet: Murine orthologs of human genes downregulated in orthochromatophilic erythroblasts are repressed by RB1. The line charts show human RNA sequencing data gene normalized by Z-score and organized by k-means clustering into 6 clusters. Eight sorted human populations are shown: CD34+, BFU-E, CFU-E, proerythroblast (PRO), early basophilic erythroblast (E-BASO), late basophilic erythroblast (L-BASO), polychromatophilic erythroblast (POLY), and orthochromatophilic erythroblast (ORTHO). The bar graphs show the distribution of the murine orthologs of the genes in each cluster by bin. The color scheme is consistent with other figures.

Article Snippet: We used the following primary antibodies: RB1 (554136), Ter119 (553670), N-terminal p130 (610262) from BD Transduction Labs; p107 and C-terminal p130 (C-20) from Santa Cruz; β-actin (A5441) from Sigma.

Techniques: RNA Sequencing Assay

Journal: Cell Cycle

Article Title: Repression by RB1 characterizes genes involved in the penultimate stage of erythroid development

doi: 10.1080/15384101.2015.1090067

Figure Lengend Snippet: Ser/Arg-rich splicing factors are repressed by RB1 during terminal erythroid differentiation. Heat map of Ser/Arg-rich splicing factor expression in control and RB1-deficient FLC in culture at 0 hours, 24 hours, and 48 hours. Each row corresponds to a unique probe set. Z-scores are gene normalized over the time course (using control and RB1 deleted samples).

Article Snippet: We used the following primary antibodies: RB1 (554136), Ter119 (553670), N-terminal p130 (610262) from BD Transduction Labs; p107 and C-terminal p130 (C-20) from Santa Cruz; β-actin (A5441) from Sigma.

Techniques: Expressing

Journal: Cell reports

Article Title: The Lineage Determining Factor GRHL2 Collaborates with FOXA1 to Establish a Targetable Pathway in Endocrine Therapy-Resistant Breast Cancer

doi: 10.1016/j.celrep.2019.09.032

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Polyclonal Anti-H3K27Ac , Diagenode , Cat#C15410196; RRID:AB_2637079.

Techniques: Microarray, Recombinant, cDNA Synthesis, SYBR Green Assay, Sample Prep, ChIP-sequencing, Negative Control, Library Amplification, Software, Cell Culture, Membrane, Magnetic Beads, DNA Purification, Protease Inhibitor