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: Nature Communications

Article Title: CTCF-mediated chromatin looping in EGR2 regulation and SUZ12 recruitment critical for peripheral myelination and repair

doi: 10.1038/s41467-020-17955-2

Figure Lengend Snippet: a Western blots for CTCF, MBP, MPZ, and EGR2 in proliferating and differentiated rat SC cultures. GAPDH served as a loading control. n = 2 independent experiments. b Relative qPCR expression of Ctcf , Mbp , Mpz , and Egr2 in proliferating and differentiated rat SC cultures. Data are presented as means ± SEM., *** P < 0.001, n = 3 independent experiments; two-tailed unpaired Student’s t -test, P ( Ctcf) = 0.00021, P ( Mbp) = 2.8E-05, P ( Mpz) = 1.7E-06, P ( Egr2) = 3.9E-05. c Colocalization of CTCF with SOX10 in SC nuclei from mice at P7, P14, and P62 evaluated by immunofluorescence labeling. Representative images are shown. n = 3 nerve tissues at each time point. Arrows indicate SOX10 + /CTCF + SCs; arrowheads indicate SOX10 + /CTCF − SCs. Scale bars: 50 μm. d The percentage of CTCF + nuclei in SCs (SOX10 + ) in sciatic nerves from P7, P14, and P62 mice. n = 3 control tissues at each time point. Data are presented as means ± SEM., * P < 0.05, ** P < 0.01; n = 3 nerve tissues at each time point; one-way ANOVA with multiple comparisons test. P (P14) = 0.0392, P (P62) = 0.0052. e Relative qPCR expression of Ctcf in mouse sciatic nerves at various developmental stages. Data are presented as means ± SEM., ** P < 0.01, *** P < 0.001; n = 3 nerve tissues at each time point; one-way ANOVA with multiple comparisons test, P (P7) = 0.0067, P (P10) = 0.0004, P (P21) = 0.1503, P (P60) = 0.0077. Source data are provided as a Source Data file.

Article Snippet: We used antibodies against CTCF (rabbit, Cell Signaling Technology, 3417 S, 1:1000), MBP (goat, Santa Cruz Biotechnology, sc-13914, 1:500), MPZ (rabbit, Abcam, ab31851, 1:1000), KROX20/EGR2 (rabbit, Santa Cruz Biotechnology, sc-20690, 1:200 or Guinea pig, provided by Michael Wegner, 1:1000), SUZ12 (rabbit, Cell Signaling Technology, 3737 S, 1:1000), EED (rabbit, Millipore, 17-10034, 1:1000), EZH2 (rabbit, Cell Signaling Technology, 5246 P, 1:1000), H3 (rabbit, Cell Signaling Technology, 4499 S, 1:1000), H3K27me3 (rabbit, Cell Signaling Technology, 9733 S, 1:1000), H3K27me2/3 (mouse, Active motif, 39536, 1:1000), H3K36me3 (rabbit, Abcam, ab9050, 1:1000), H3K4me1 (mouse, Active motif, 39635, 1:1000), H3K27ac (rabbit, Cell Signaling Technology, 4353 S, 1:1000), and GAPDH (mouse, Millipore, MAB374, 1:5000).

Techniques: Western Blot, Control, Expressing, Two Tailed Test, Immunofluorescence, Labeling

Journal: Nature Communications

Article Title: CTCF-mediated chromatin looping in EGR2 regulation and SUZ12 recruitment critical for peripheral myelination and repair

doi: 10.1038/s41467-020-17955-2

Figure Lengend Snippet: a qRT-PCR analysis of Ctcf , Sox10 , Egr2 , and Mpz expression in rat SCs transfected with control nontargeting siRNA and si Ctcf for 24 h and induced to differentiate for 9 h. n = 3 independent experiments, P ( Ctcf ) = 3.03E-05, P ( Sox10 ) = 0.0433, P ( Egr2 ) = 0.000107, P ( Mpz ) = 0.000293. b–d Rat SCs were transfected with control siRNA or si Ctcf for 24 h and induced to differentiate for 9 h and CTCF- ( b ), EGR2- and OCT6-positive ( c ) cells were visualized by immunofluorescence microscopy and d quantified; n = 3 independent experiments. Arrows indicate CTCF + or EGR2 + /OCT6 + SCs. Scale bars: 50 µm. n = 3 independent experiments, P (EGR2) = 0.00069, P (OCT6) = 0.99. e Western blots for CTCF and EGR2 in co-cultures of rat DRGs and SCs treated with control siRNA or si Ctcf . GAPDH served as a loading control. n = 4 independent experiments. f Rat SCs treated with control siRNA or si Ctcf were seeded onto rat DRGs. After 10 days, co-cultures were immunostained for MBP and neurofilament-M. Images are representative of n = 4 independent experiments. Scale bars: 100 μm. g Quantification of the number of MBP + segments per mm 2 of area in myelinating co-cultures of DRGs and SCs treated with control siRNA or si Ctcf . n = 4 independent experiments, P = 0.0068. h Western blots for CTCF in rat Schwann cells induced to differentiate following transfection with control or CTCF expression vectors. n = 2 independent experiments. i qRT-PCR quantification of differentiation regulators and negative regulators in rat SCs induced to differentiate following transfection with control or CTCF expression vectors. n = 3 independent experiments, P ( Egr2 ) = 0.0012, P ( Cnp ) = 0.00068, P ( Mbp ) = 0.011, P ( Mpz ) = 2.9E-05, P ( Pmp22 ) = 6.7E-05, P ( Sox2 ) = 0.00026, P ( Hes1 ) = 0.028, P ( Mki67 ) = 0.00024. Data are presented as means ± SEM., * P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed unpaired Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: We used antibodies against CTCF (rabbit, Cell Signaling Technology, 3417 S, 1:1000), MBP (goat, Santa Cruz Biotechnology, sc-13914, 1:500), MPZ (rabbit, Abcam, ab31851, 1:1000), KROX20/EGR2 (rabbit, Santa Cruz Biotechnology, sc-20690, 1:200 or Guinea pig, provided by Michael Wegner, 1:1000), SUZ12 (rabbit, Cell Signaling Technology, 3737 S, 1:1000), EED (rabbit, Millipore, 17-10034, 1:1000), EZH2 (rabbit, Cell Signaling Technology, 5246 P, 1:1000), H3 (rabbit, Cell Signaling Technology, 4499 S, 1:1000), H3K27me3 (rabbit, Cell Signaling Technology, 9733 S, 1:1000), H3K27me2/3 (mouse, Active motif, 39536, 1:1000), H3K36me3 (rabbit, Abcam, ab9050, 1:1000), H3K4me1 (mouse, Active motif, 39635, 1:1000), H3K27ac (rabbit, Cell Signaling Technology, 4353 S, 1:1000), and GAPDH (mouse, Millipore, MAB374, 1:5000).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Immunofluorescence, Microscopy, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: CTCF-mediated chromatin looping in EGR2 regulation and SUZ12 recruitment critical for peripheral myelination and repair

doi: 10.1038/s41467-020-17955-2

Figure Lengend Snippet: a Excised exon 8 of the floxed Ctcf allele by Dhh-Cre . b Co-labeling of CTCF with SOX10 in control and mutant sciatic nerves at P7 ( n = 3 animals/genotype). Arrows indicate SOX10 + /CTCF + SCs. Scale bars: 50 μm. c The percentage of CTCF + nuclei in SCs (SOX10 + ) from control and Ctcf cKO sciatic nerves at P7. n = 3 animals/genotype, P = 1.73E-05. d Survival curves of control and Ctcf cKO mice. n = 25 for control and n = 23 for Ctcf cKO mice, *** P < 0.001. e Representative photographs of sciatic nerves from P13 control and Ctcf cKO mice. n = 3 animals/genotype. f Immunofluorescence labeling of MBP (red) in P7 control and Ctcf cKO sciatic nerves. n = 3 animals/genotype. Scale bars: 50 μm. g The mRNA levels of myelin-related genes in P7 control and Ctcf cKO sciatic nerves. n = 6 animals/genotype. P ( Prx ) = 1.9E-08, P ( Mbp ) = 2.0E-08, P ( Mpz ) = 8.5E-09. h, i Ultrastructure of control and Ctcf cKO sciatic nerves at ( h ) P1 and P7 and at ( i ) 8 weeks. n = 3 animals/genotype. Arrows and arrowheads indicate immature SCs and unsorted axons, respectively. Scale bars: 4 μm. j A diagram showing the tamoxifen (TAM) administration scheme. k Immunofluorescent labeling of CTCF (green) nuclei in control and Ctcf iKO sciatic nerves at P14. Scale bars: 50 μm. n = 3 animals/genotype. l EM images of P14 sciatic nerves from control and Ctcf iKO mice. n = 4 animals/genotype. Arrow indicates myelin membrane. Scale bars: 4 μm, and 1 μm in the inset on the right panel. m Myelinated axon numbers 10 −4 μm −2 sections of P14 sciatic nerves from control and Ctcf iKO mice. n = 4 animals/genotype, P = 0.0006. Data are presented as means ± SEM., *** P < 0.001; Statistical analyses performed using two-tailed unpaired Student’s t -test; Log-rank test used for survival curve. Source data are provided as a Source Data file.

Article Snippet: We used antibodies against CTCF (rabbit, Cell Signaling Technology, 3417 S, 1:1000), MBP (goat, Santa Cruz Biotechnology, sc-13914, 1:500), MPZ (rabbit, Abcam, ab31851, 1:1000), KROX20/EGR2 (rabbit, Santa Cruz Biotechnology, sc-20690, 1:200 or Guinea pig, provided by Michael Wegner, 1:1000), SUZ12 (rabbit, Cell Signaling Technology, 3737 S, 1:1000), EED (rabbit, Millipore, 17-10034, 1:1000), EZH2 (rabbit, Cell Signaling Technology, 5246 P, 1:1000), H3 (rabbit, Cell Signaling Technology, 4499 S, 1:1000), H3K27me3 (rabbit, Cell Signaling Technology, 9733 S, 1:1000), H3K27me2/3 (mouse, Active motif, 39536, 1:1000), H3K36me3 (rabbit, Abcam, ab9050, 1:1000), H3K4me1 (mouse, Active motif, 39635, 1:1000), H3K27ac (rabbit, Cell Signaling Technology, 4353 S, 1:1000), and GAPDH (mouse, Millipore, MAB374, 1:5000).

Techniques: Labeling, Control, Mutagenesis, Immunofluorescence, Membrane, Two Tailed Test

Journal: Nature Communications

Article Title: CTCF-mediated chromatin looping in EGR2 regulation and SUZ12 recruitment critical for peripheral myelination and repair

doi: 10.1038/s41467-020-17955-2

Figure Lengend Snippet: a Volcano plot of transcriptome profiles of control and Ctcf cKO sciatic nerves ( n = 2 animals/genotype). Red and blue dots represent significantly downregulated and upregulated genes in Ctcf cKO nerves compared to the control, respectively ( P < 0.05, fold-change > 1.5). b Heatmap of representative genes and their categories differentially expressed in control and Ctcf cKO sciatic nerves ( n = 2 animals/genotype). c , d Bar plots of gene ontology analysis of genes c downregulated and d upregulated genes in Ctcf cKO sciatic nerves compared with control nerves. Each dot (connected by lines) represents the gene count of the corresponding biological function categories. n = 2 independent tissues/genotype. e qPCR analysis of genes related to SC development that are decreased (left) and increased (right) in Ctcf cKO sciatic nerves relative to control. f GSEA enrichment scores for myelin sheath (left) and lipid biosynthetic process (right) gene sets in control and Ctcf cKO sciatic nerves. g GSEA enrichment scores for cell-cycle gene sets in control and Ctcf cKO sciatic nerves. Data are presented as means ± SEM., *** P < 0.001, ** P < 0.01, * P < 0.05, n = 3 animals/genotype; two-tailed unpaired Student’s t -test, P (Prx) = 2.6e-05, P (Mbp) = 4.9E-05, P (Mpz) = 5.3E-06, P (Hmgcr) = 0.0014, P (Egr2) = 8.6E-05, P (Itgb1) = 0.008, P (Itgb3bp) = 0.00022, P (Itgb5) = 0.0021, P (Itgb8) = 0.00017, P (Ccnd1) = 7.4E-05, P (Ccng1) = 5.1E-05, P (Ccno) = 0.0004, P (Cdc7) = 6.4E-05, P (Cdk5r2) = 1.6E-05, P (Ccnb1) = 3.2E-05, P (Notch1) = 0.00102, P (Hes5) = 0.028, P (Id2) = 0.23, P (Id4) = 3.3E-05. Source data are provided as a Source Data file.

Article Snippet: We used antibodies against CTCF (rabbit, Cell Signaling Technology, 3417 S, 1:1000), MBP (goat, Santa Cruz Biotechnology, sc-13914, 1:500), MPZ (rabbit, Abcam, ab31851, 1:1000), KROX20/EGR2 (rabbit, Santa Cruz Biotechnology, sc-20690, 1:200 or Guinea pig, provided by Michael Wegner, 1:1000), SUZ12 (rabbit, Cell Signaling Technology, 3737 S, 1:1000), EED (rabbit, Millipore, 17-10034, 1:1000), EZH2 (rabbit, Cell Signaling Technology, 5246 P, 1:1000), H3 (rabbit, Cell Signaling Technology, 4499 S, 1:1000), H3K27me3 (rabbit, Cell Signaling Technology, 9733 S, 1:1000), H3K27me2/3 (mouse, Active motif, 39536, 1:1000), H3K36me3 (rabbit, Abcam, ab9050, 1:1000), H3K4me1 (mouse, Active motif, 39635, 1:1000), H3K27ac (rabbit, Cell Signaling Technology, 4353 S, 1:1000), and GAPDH (mouse, Millipore, MAB374, 1:5000).

Techniques: Control, Two Tailed Test

Journal: Toxics

Article Title: Mono-(2-ethylhexyl) Phthalate (MEHP)-Induced Telomere Structure and Function Disorder Mediates Cell Cycle Dysregulation and Apoptosis via c-Myc and Its Upstream Transcription Factors in a Mouse Spermatogonia-Derived (GC-1) Cell Line

doi: 10.3390/toxics11050448

Figure Lengend Snippet: MEHP inhibits c-Myc transcriptional regulators in GC-1 cells. ( A ) The Cistrome DB, TRRUST, and Genecards databases were screened for c-Myc upstream transcription factors and the results of the unique and common parts between each group were visualized using Venn diagrams. ( B ) GC-1 cells were exposed to different concentrations of MEHP for 48 h, and the mRNA expression levels of Ctcf , Stat3 , Esr1 , C-jun , and Foxa1 were detected by RT-qPCR. Beta-actin ( β-actin ) was used as the housekeeper gene. ( C ) After 48 h of MEHP treatment, the relative protein levels of CTCF, STAT3, ESR1, C-JUN, and FOXA1 in the GC-1 cells were detected by Western blot, and the expression levels of these target proteins relative to β-actin were quantified by densitometric analysis of the bands. The results are expressed as the mean ± SD, n ≥ 3. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the control group treated with DMSO.

Article Snippet: The CTCF (A19588), STAT3 (A19566), and ESR1 (A12976) antibodies were from ABclonal Technology (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Developmental biology

Article Title: Trim33 is required for appropriate development of pre-cardiogenic mesoderm

doi: 10.1016/j.ydbio.2019.03.018

Figure Lengend Snippet: A, heat map showing Trim33 ChIP-Seq read density around the merged peak regions. B, signal intensity plot representing Trim33 and input ChIP-Seq profiles around the merged peak regions. C, pie chart displaying distributions of Trim33 peaks across promoter, intragenic and intergenic regions. D, functional annotation analysis using GREAT reveals that Trim33 preferentially binds to genes associated with stem cell maintenance and mesoderm formation (GO: Biological Process), and holoprosencephaly, VSD and craniosynostosis (GO: Disease Ontology (developmental diseases)). E, three most common consensus motifs recognized by Trim33 are Ctcf, Tead and Isl1 motifs. F, venn diagram showing the overlap of Trim33, Ctcf and H3K27ac target genes as identified by ChIP-Seq. G, examples of genome browser images depicting different Trim33, Ctcf and H3K27ac profiles. H, ChIP-qPCR at enriched regions found near the four indicated genes; IP with the anti-Trim33 antibodies (controls, closed circles; mutants, open circles; n = 5 for Cbfa2t2l; n = 3 for Ctgf, Gse1 and Phc2; *,p < 0.05). I, co-immunoprecipitation of Trim33 with Ctcf and Smad2 in EBs transfected with Flag-tagged Trim33 cDNA and un-tagged Ctcf cDNAs with or without Activin stimulation (100 ng/ml, 40 min); n = 3.

Article Snippet: Transfection, immunoprecipitation and western blot assays EBs at day 6 of differentiation were dissociated with Trypsin, plated and transfected with full-length Myc/Flag-tagged mTrim33 cDNA (Origene MR227454) and full-length mCtcf cDNA (Origene MC202667) using ViaFect Transfection reagent (Promega PRE4981).

Techniques: ChIP-sequencing, Functional Assay, ChIP-qPCR, Immunoprecipitation, Transfection