Journal: Molecular and Cellular Biology

Article Title: LDB1 Enforces Stability on Direct and Indirect Oncoprotein Partners in Leukemia

doi: 10.1128/MCB.00652-19

Figure Lengend Snippet: Halo-tagged proteins expressed from recombinant lentiviruses have physiological abundance and localization and can be precisely quantified in lysates or live cells. (A) Cell lysates were prepared from untransduced Jurkat cells; Jurkat cells transduced with Halo-LMO2; untransduced K562 cells; K562 cells transduced with Halo-LMO2; and KOPT-K1, LOUCY, and U937 cells. Lysates were subjected to 4% to 15% SDS-PAGE, transferred, and blotted with anti-LDB1, anti-LMO2 monoclonal antibody, anti-Halo, anti-TAL1, and anti-VCP (gel loading control). Molecular weight standards were run on the same gel and are shown at the right. (B) The top panel shows an immunoblot of lysates prepared from U2OS with a Halo knock-in at CTCF (lane 1) or lysates from Jurkat cells transduced with the constructs shown in the grid above (lanes 2 to 14). Empty vector control is shown as EBFPII-Hygro (lane 2). Lanes 2 to 9 show lysates from Jurkat cells lentivirally expressing Halo-CTCF at increasing MOIs. Lanes 10 to 14 show lysates from Jurkat cells lentivirally expressing Halo-SOX2 at increasing MOIs. The top panel shows gradient SDS-PAGE, transfer, and blotting with anti-CTCF antibody. The middle panel shows direct in-gel Halo fluorescence. Live cells prepared as described for panel A were labeled with cell-permeative fluorescent ligand R110, lysed, and subjected to gradient SDS-PAGE. The gel was visualized for green fluorescence as described in Materials and Methods. (C) Live Jurkat cells expressing Halo-tagged proteins were labeled with R110 Halo ligand, washed, and subjected to flow cytometry. Histograms show FITC fluorescence of the various cells in comparison to the labeled U2OS cells, which have a Halo tag knocked into the CTCF gene. (D) Table showing calculated copy numbers of Halo-tagged proteins based on the absolute values derived from the Halo knock-in cell line. Lentiviral Halo CTCF and Halo SOX2 were quantified from cell populations infected at intermediate and low MOIs, respectively. (E) Confocal microscopy of Jurkat cells lentivirally expressing Halo-LMO2 and labeled with Halo ligand, R110, and nuclear stain (Syto 17 Red). The right panel shows a merged image. Voxel quantification showed that 95% of Halo-LMO2 was nuclear.

Article Snippet: Catalog no. S7579 pBluescript SK Stratagene Iscove's modified Dulbecco's medium (IMDM) Gibco Catalog no. 12200-036 RPMI 1640 Gibco Catalog no. 31800-022 Penicillin-streptomycin solution 10× Corning Catalog no. 30-022-CI Geneticin Gibco Catalog no. 10131-027 0.05% trypsin, 0.53 mM EDTA 1× [−]sodium bicarbonate Corning Catalog no. 20116004 Puromycin dihydrochloride Fisher Bioreagents Catalog no. BP2956-100 Pierce protease inhibitor tablets Thermo Scientific Catalog no. A32965 Hygromycin B-PBS (50 mg/ml) Invitrogen Catalog no. 10687010 Anti-FLAG M2 resin Sigma Catalog no. A2220 Protein A/G resin Santa Cruz Polyvinylidene difluoride (PVDF) membrane GE Catalog no. 10600022 SuperSignal PicoWest Plus Thermo/Pierce Catalog no. 1863099 Experimental models: cell lines Human: HEK 293 ATCC Human: Jurkat ATCC Human: K562 ATCC Human: KOPTK1 ATCC Human: LOUCY ATCC Human: U937 ATCC Human: U2OS C32 Halo-CTCF Hansen et al. ( 27 ) U2OS endogenous knock-in cell line where all endogenous copies of CTCF have been N-terminally tagged with FLAG-HaloTag; clone 32 Software and algorithms Flowjo 10.3 analysis software FlowJo, LLC Contact corresponding author for URL Ideas software Amnis Corporation Contact corresponding author for URL ImageLab 5.2.1 BioRad Contact corresponding author for URL Imaris Bitplane Inc. Other CytoFLEX benchtop cytometer Beckman Contact corresponding author for URL Leica TCS SP8 confocal imaging system Leica Contact corresponding author for URL ImageStream Mk II Amnis Contact corresponding author for URL Open in a separate window Reagents and resources Experimental model and subject details. (i) Cell lines.

Techniques: Recombinant, Transduction, SDS Page, Control, Molecular Weight, Western Blot, Knock-In, Construct, Plasmid Preparation, Expressing, Fluorescence, Labeling, Flow Cytometry, Comparison, Derivative Assay, Infection, Confocal Microscopy, Staining

Journal: Molecular and Cellular Biology

Article Title: LDB1 Enforces Stability on Direct and Indirect Oncoprotein Partners in Leukemia

doi: 10.1128/MCB.00652-19

Figure Lengend Snippet: Reagents and resources

Article Snippet: Catalog no. S7579 pBluescript SK Stratagene Iscove's modified Dulbecco's medium (IMDM) Gibco Catalog no. 12200-036 RPMI 1640 Gibco Catalog no. 31800-022 Penicillin-streptomycin solution 10× Corning Catalog no. 30-022-CI Geneticin Gibco Catalog no. 10131-027 0.05% trypsin, 0.53 mM EDTA 1× [−]sodium bicarbonate Corning Catalog no. 20116004 Puromycin dihydrochloride Fisher Bioreagents Catalog no. BP2956-100 Pierce protease inhibitor tablets Thermo Scientific Catalog no. A32965 Hygromycin B-PBS (50 mg/ml) Invitrogen Catalog no. 10687010 Anti-FLAG M2 resin Sigma Catalog no. A2220 Protein A/G resin Santa Cruz Polyvinylidene difluoride (PVDF) membrane GE Catalog no. 10600022 SuperSignal PicoWest Plus Thermo/Pierce Catalog no. 1863099 Experimental models: cell lines Human: HEK 293 ATCC Human: Jurkat ATCC Human: K562 ATCC Human: KOPTK1 ATCC Human: LOUCY ATCC Human: U937 ATCC Human: U2OS C32 Halo-CTCF Hansen et al. ( 27 ) U2OS endogenous knock-in cell line where all endogenous copies of CTCF have been N-terminally tagged with FLAG-HaloTag; clone 32 Software and algorithms Flowjo 10.3 analysis software FlowJo, LLC Contact corresponding author for URL Ideas software Amnis Corporation Contact corresponding author for URL ImageLab 5.2.1 BioRad Contact corresponding author for URL Imaris Bitplane Inc. Other CytoFLEX benchtop cytometer Beckman Contact corresponding author for URL Leica TCS SP8 confocal imaging system Leica Contact corresponding author for URL ImageStream Mk II Amnis Contact corresponding author for URL Open in a separate window Reagents and resources Experimental model and subject details. (i) Cell lines.

Techniques: Recombinant, Staining, Modification, Protease Inhibitor, Membrane, Knock-In, Software, Cytometry, Imaging

Journal: Oncogene

Article Title: Genome-wide screen for differentially methylated long noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as regulated by enhancer DNA methylation with prognostic relevance for human breast cancer

doi: 10.1038/onc.2017.246

Figure Lengend Snippet: Candidate lncRNAs with differentially methylated promoters and neighboring protein coding genes

Article Snippet: Electrophoresis, blotting, detection and quantification were performed as described in Pappa et al. For detection of Esrp2, anti-Esrp2 monoclonal antibody (210-301-C32S, Rockland Immunochemicals, USA) was used at 1:250 dilution.

Techniques: Methylation, Expressing

Journal: Oncogene

Article Title: Genome-wide screen for differentially methylated long noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as regulated by enhancer DNA methylation with prognostic relevance for human breast cancer

doi: 10.1038/onc.2017.246

Figure Lengend Snippet: Esrp2 and Esrp2-as are coordinately overexpressed in C3(1) mammary gland tumors. ( a ) Genomic organization of Esrp2 (dark blue) and Esrp2-as variants 1-4 (v1-4, light blue). Location of primers detecting Esrp2-as v1+2 or v1-4, respectively, is indicated in red. ( b ) Relative expression levels as determined by RT–qPCR are significantly different between tumor ( n =11) and normal samples ( n =9) for Esrp2 and Esrp2-as (v1-4), but not for Esrp2-as (v1+2). Samples were derived from animals aged 20-24 weeks, and expression levels were normalized to three reference genes ( Hprt1, Tbp, β-Actin ). Mann-Whitney U test, ** P <0.01, *** P <0.001. ( c ) Esrp2 and Esrp2-as v1-4 expression assessed by RT–qPCR highly correlates in tumors, normal mammary gland, cell lines, spleen, and liver samples. Spearman’s rank correlation coefficient rho=0.88 ( P <0.0001).

Article Snippet: Electrophoresis, blotting, detection and quantification were performed as described in Pappa et al. For detection of Esrp2, anti-Esrp2 monoclonal antibody (210-301-C32S, Rockland Immunochemicals, USA) was used at 1:250 dilution.

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, MANN-WHITNEY

Journal: Oncogene

Article Title: Genome-wide screen for differentially methylated long noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as regulated by enhancer DNA methylation with prognostic relevance for human breast cancer

doi: 10.1038/onc.2017.246

Figure Lengend Snippet: DNA methylation levels inversely correlate with expression levels of Esrp2 and Esrp2-as . ( a ) Upper : genomic organization of Esrp2 (dark blue) and Esrp2-as variants 1-4 (v1-4, light blue) and the CGI overlapping the Esrp2 TSS (green). Positions of EPITYPER Amplicons A1-A14 are indicated by horizontal black bars, covering the DMR (pink), the CGI, and CGI shores on both sides. Lower : MCIp-seq detection of methylated DNA fragments in tumors (red) and normal WT mammary glands (blue) of animals at 20 and 24 weeks of age. Each lane represents average reads obtained for three individual samples. ( b , c ) Heatmap of DNA methylation levels in tumor samples ( n =11) and normal mammary gland tissue ( n =9) ( b ), or of various murine cell lines ( c ), with each row representing one individual sample and each column one CpG unit comprising of 1 to 4 individual CpG sites. Methylation levels are depicted by a color-coded gradient from 0% (light yellow) to 100% methylation (blue). Gray squares indicate failed measurements. ( d , e ) Correlation between average methylation of amplicons A1-A14 and Esrp2/Esrp2-as expression levels normalized to three reference genes, calculated by Spearman’s rank correlation for tumor/normal tissues ( d ) and cell lines ( e ). * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Electrophoresis, blotting, detection and quantification were performed as described in Pappa et al. For detection of Esrp2, anti-Esrp2 monoclonal antibody (210-301-C32S, Rockland Immunochemicals, USA) was used at 1:250 dilution.

Techniques: DNA Methylation Assay, Expressing, Methylation

Journal: Oncogene

Article Title: Genome-wide screen for differentially methylated long noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as regulated by enhancer DNA methylation with prognostic relevance for human breast cancer

doi: 10.1038/onc.2017.246

Figure Lengend Snippet: Esrp2 and Esrp2-as are coordinately expressed from a bidirectional promoter and regulated by methylation of a proximal enhancer. ( a ) Treatment of M27 H4 cells with 1 μ m DAC for 72 h with daily renewal decreases DNA methylation levels. Depicted is the mean methylation±SD for amplicons A1-A14 of three independent experiments. Mann-Whitney U test (one-sided), * P <0.05. ( b ) DAC treatment induces reexpression of Esrp2 and Esrp2-as v1-4 in M27 H4 cells. Depicted is the mean expression±SD normalized to DMSO solvent control of three independent experiments. Unpaired Student’s t-test (one-sided), *** P <0.001, NS, not significant. ( c ) Schematic representation of luciferase reporter constructs to determine promoter and enhancer activity of fragments covering the Esrp2 region. Location of Esrp2 and Esrp2-as are depicted in gray and promoter constructs are marked in red and green. Enhancer reporters are displayed by blue boxes combined with a minimal promoter (purple). ( d , e ) Reporter plasmids with firefly luciferase for promoter ( d ) and bidirectional promoter ( e ) activity were transiently transfected into Hepa1.6 cells and luciferase activity was measured 48 h post transfection and normalized to a co-transfected CMV- Renilla luciferase construct. Transfections were conducted parallel in 8 technical replicates and reported is the mean±SEM of four independent experiments normalized to the pGL4.10 EV. ( f ) Reporter constructs for enhancer activity. Transfections and measurements were conducted and reported as in ( d ) and normalized to the pGL4.23 EV. Unpaired Student’s t-test (two-sided), asterisks represent comparisons against EV. *P <0.05, ** P <0.01. RLA, relative luciferase activity.

Article Snippet: Electrophoresis, blotting, detection and quantification were performed as described in Pappa et al. For detection of Esrp2, anti-Esrp2 monoclonal antibody (210-301-C32S, Rockland Immunochemicals, USA) was used at 1:250 dilution.

Techniques: Methylation, DNA Methylation Assay, MANN-WHITNEY, Expressing, Solvent, Control, Luciferase, Construct, Activity Assay, Transfection

Journal: Oncogene

Article Title: Genome-wide screen for differentially methylated long noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as regulated by enhancer DNA methylation with prognostic relevance for human breast cancer

doi: 10.1038/onc.2017.246

Figure Lengend Snippet: Knockdown of Esrp2-as reduces Esrp2 protein levels, globally alters expression of genes involved in cellular motility and inhibits cell proliferation. ( a ) Knockdown of Esrp2-as by Locked Nucleic Acid antisense Gapmers (LNAs) in the M6 cell line for 72 h. Expression levels were assessed by RNA-seq; CPM values are normalized to cells transfected with negative control LNA. Mean±SD of two independent experiments. Statistical significance was assessed using Student’s t-test with * P <0.05. ( b ) M27 H4 cells were transiently transfected with constructs containing Esrp2-as v4. Expression levels 72 h post-transfection were assessed by RNA-seq; CPM values are normalized to cells transfected with the empty vector (EV). Mean±s.d. of three independent experiments. Statistical significance was assessed using Student’s t -test with Welch’s correction ( P =0.116, NS). ( c ) Esrp2 protein expression in the M6 cell line 72 h after knockdown of Esrp2-as with LNA#2. M27 H4 cells stably overexpressing Esrp2 were used as a positive control (pos. ctrl). Band intensities were normalized to β-actin levels and are semi-quantitatively evaluated relative to the negative control using TINA v2.09. Depicted is the mean±SD of three independent experiments. Statistical significance was assessed using a one-sample t-test with the neg. control set to 1.0, with *P <0.05. ( d ) Heatmap of top 40 up- and top 20 downregulated genes after Esrp2-as knockdown in M6 cells. Depicted are log2 CPM values of two biological replicates. ( e ) Overlap of differentially expressed genes after knockdown of Esrp2-as with gene sets in MSigDB using GSEA. ( f ) Cell proliferation was assessed by SRB staining on five consecutive days starting 24 h post transfection (day 0). Cell proliferation was adjusted for the number of seeded cells and is depicted in relation to the negative control or EV at day 5 (set as 100%). Shown is the mean±SD of three independent experiments (represented by black dots). Statistical significance was assessed using the one-sample t -test with ** P <0.01, NS, not significant.

Article Snippet: Electrophoresis, blotting, detection and quantification were performed as described in Pappa et al. For detection of Esrp2, anti-Esrp2 monoclonal antibody (210-301-C32S, Rockland Immunochemicals, USA) was used at 1:250 dilution.

Techniques: Knockdown, Expressing, RNA Sequencing, Transfection, Negative Control, Construct, Plasmid Preparation, Stable Transfection, Positive Control, Control, Staining

Journal: Oncogene

Article Title: Genome-wide screen for differentially methylated long noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as regulated by enhancer DNA methylation with prognostic relevance for human breast cancer

doi: 10.1038/onc.2017.246

Figure Lengend Snippet: A human homolog of Esrp2-as is overexpressed in human breast cancer and associated with poor prognosis. ( a ) UCSC Genome Browser scheme of the human ESPR2 locus on chr16 depicts the genomic location of the human ESRP2 gene (blue), CpG islands (green), genomic location of ESRP2-AS (red), MCF-7 CAGE-seq peaks for the positive (red) and negative strand (red), positions of RT–qPCR primers to quantify expression of ESRP2-AS (RNA1-4, red), strand-specific RNA-seq signals obtained with MCF7 whole cell lysates, cytosolic and nuclear fraction (black), location of mouse EPITYPER MassArray amplicons (using UCSC genome browser liftover tool, beige), DNA methylation levels for individual CpG sites (indicated by beige lines) assessed by WGBS (downloaded from TCGA), CpG sites covered on the Illumina 450 k methylation array (blue). ( b ) Left: Relative expression of ESRP2-AS (using four primer pairs designated as RNA1-4) and ESRP2 in MCF7 ( n =2), HepG2 ( n =1), MDA-MB231 ( n =1) and MCF10a ( n =1) human (cancer) cell lines, assessed by RT–qPCR. Right: Fold change of ESRP2 and ESRP2-AS levels in MCF7 ( n =2) and MDA-MB231 cells ( n =1) after DAC treatment, relative to DMSO solvent control set as 1. Statistical significance was assessed using the one-sample t-test with * P <0.05. ( c ) ESRP2 (left) and ESRP2-AS (middle) expression in human breast cancer samples from the TCGA BRCA breast invasive carcinoma dataset. RNA-seq data (log2 normalized read counts) for 1002 tumor samples were compared to 45 normal control samples by two-sided Student’s t -test with **** P <0.0001, * P <0.05. Right: Spearman correlation of ESPR2 and ESRP2-as levels (log2 norm. read counts) in normal breast tissue (blue) and tumor tissue (red) in the TCGA BRCA dataset. ( d ) Kaplan–Meier curves of disease-free survival are plotted for ESRP2 and ESRP2-AS expression. A total of 814 informative breast cancer cases from TCGA BRCA with an overall number of 107 events are separated by expression levels into a low (black) and high (red) expressing group. P -values for log-rank Mantel-Cox test; HR (hazard ratio) with 95% confidence interval indicated in parenthesis.

Article Snippet: Electrophoresis, blotting, detection and quantification were performed as described in Pappa et al. For detection of Esrp2, anti-Esrp2 monoclonal antibody (210-301-C32S, Rockland Immunochemicals, USA) was used at 1:250 dilution.

Techniques: Quantitative RT-PCR, Expressing, RNA Sequencing, DNA Methylation Assay, Methylation, Solvent, Control