Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) E17.5-E18.5 cortices were dissociated and plated on poly-D-lysine. After 10d, cultures were stained for pan neuronal (MAP2), astrocyte (GFAP) and microglia (IBA1) markers, and cell type composition was determined by quantitative analysis of immunofluorescence images. Based on 6 Rad21 +/+ Nex Cre and 8 Rad21 lox/lox Nex Cre different samples analysed in 4 independent experiments. b) Immunofluorescence staining of Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre neuronal explant cultures for RAD21 and MAP2 (left) and distribution of RAD21 expression by MAP + neurons (right). Note the discontinuous distribution of RAD21 expression in Rad21 lox/lox Nex Cre neurons. Three independent experiments per genotype. DAPI marks nuclei. Scale bar = 60 μm. c) Immunofluorescence staining for RAD21, MAP2, and the marker of GABAergic inhibitory neurons, GAD67 (left). Distribution of RAD21 expression in GAD67 + and GAD67 - neurons (right). Note that the discontinuous distribution of RAD21 expression in Rad21 lox/lox Nex Cre neuronal explant cultures is due to GAD67 + GABAergic inhibitory neurons. Three independent experiments for Rad21 +/+ Nex Cre and 6 independent experiments for Rad21 lox/lox Nex Cre . DAPI marks nuclei. Scale bar = 20 μm. d) Quantitative RT-PCR analysis of Rad21 mRNA expression in Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical explant cultures (mean ± SEM, n=18). Hprt and Ubc were used for normalization (left). RAD21 protein expression in Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical explant cultures was quantified by fluorescent immunoblots (mean ± SEM, n=6) and normalised to LaminB (center). Nex Cre RiboTag RNA-seq of analysis of Rad21 mRNA expression in Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical explant cultures (right, 3 independent biological replicates). e) 5C heat maps of a 1.72 Mb region on chromosome 2, comparing Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical explant cultures. CTCF ChIP-seq (Ref. ) and mm9 coordinates are shown for reference. Arrowheads mark the position of CTCF-based loops. Results were consistent across two replicates and 3 chromosomal regions Histograms below show the quantification of representative CTCF-based loops (arrowheads) in two independent biological replicates for control and Rad21 lox/lox Nex Cre neurons.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Staining, Immunofluorescence, Expressing, Marker, Quantitative RT-PCR, Western Blot, RNA Sequencing Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Nex Cre -dependent Rpl22-HA (RiboTag) expression is restricted to RAD21-negative cells in Rad21 lox/lox Nex Cre neurons. Immunofluorescence staining for RAD21, the pan-neuronal marker MAP2 and HA (RiboTag) in explant culture. DAPI marks nuclei. Scale bar = 40 μm. b) Nex Cre RiboTag captures excitatory neuron-specific transcripts such as Slc17a7 and Camk2a and depletes cell type-specific transcripts expressed in inhibitory neurons ( Gad1, Gad2, Slc32a1 ), astrocytes ( Gfap, Aqp4, Mlc1 ), and microglia ( Aif ). Transcript enrichment (or depletion) was calculated using the normalized counts from Nex Cre RiboTag versus standard RNA-seq in Rad21 +/+ Nex Cre neurons.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Expressing, Immunofluorescence, Staining, Marker, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Volcano plot representing log2 fold-change (FC) versus significance (-log10 of adjusted P values) of downregulated genes (1028) and upregulated genes (572) in RiboTag RNA-seq of Rad21 lox/lox Nex Cre versus Rad21 +/+ Nex Cre neurons. Red marks Rad21 . b) Analysis of gene ontology of biological functions of deregulated genes in Rad21 lox/lox Nex Cre neurons. Enrichment is calculated relative to expressed genes. c) The percentage of constitutive and activity-dependent genes deregulated in Rad21 lox/lox Nex Cre neurons in explant culture at baseline as determined by RiboTag RNA-seq. The P -value (Fisher Exact Test) and Odds ratio indicate that activity-dependent genes are more frequently deregulated than constitutive genes.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: RNA Sequencing Assay, Activity Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Examples of deregulated genes in Rad21 lox/lox Nex Cre neurons. Genes associated with autism spectrum disorders are highlighted in red. b) GSEA for downregulated genes in Nex Cre/+ Rad21 lox/lox neurons using gene sets derived from (i) KEGG pathway database, (ii) GO Biological Process Ontology, (iii) GO Molecular Function Ontology, (iv) GO Cellular Component Ontology in the Molecular Signatures Database (MSigDB). c) Overlap between human genes associated with autism spectrum disorders from the SFARI database and differentially expressed genes (left), downregulated genes (middle) and upregulated genes (right) in Rad21 lox/lox Nex Cre cortical neurons.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Expected Mendelian ratios and observed percentages of live Rad21 +/+ Nex Cre , Rad21 lox/+ Nex Cre , Rad21 lox/lox Nex Cre mice at the indicated developmental stages, n = 217. b) Immunofluorescence analysis shows neither the apoptosis marker activated caspase 3 (CC3) nor the DNA damage marker γH2AX in E16.5 (top) and E18.5 Rad21 lox/lox Nex Cre (bottom, white lines demarcate the cortex). Wild type E16.5 thymi are shown as positive controls for CC3 and γH2AX. Two biological replicates. Scale bar = 100 μm. Photomicrographs of coronal brain sections at gestational age E16 modified from the Atlas of the prenatal mouse brain are shown for orientation. c) Quantitative RT-PCR analysis of gene expression in Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre E17.5/18.5 cortical explant cultures 10 d after plating. Hprt and Ubc were used for normalization. Mean ± SEM of 3 cultures per genotype. d) Brain weights of Rad21 +/+ Nex Cre and Rad21 lox/+ Nex Cre , Rad21 lox/lox Nex Cre mice at birth (P0). Mean ± SEM of between 3 and 13 mice per genotype. e) Embryonic cortices from wild-type and Rad21 lox/lox Nex Cre mice were dissected at E17.5 and E18.5 and dissociated. Cortical cell numbers were determined by counting in Neubauer chambers. Each symbol denotes an independent experiment. Mean ± SEM are also shown.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Immunofluorescence, Marker, Modification, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Schema of cortical layers showing subplate (SP), layer 6 (VI), layer 5 (V), the cortical plate (CP), and the marginal zone (MZ). Immunofluorescence analysis of the neuronal transcription factors CUX1, TBR1, and CTIP2 at E16.5. Representative of 3 biological replicates. Scale bar = 100 μm. b) Morphology of E18.5 neurons after 1 d in explant culture. Immunofluorescence staining for the pan-neuronal marker MAP2, tubulin beta 3 (TUBB3), and DAPI. Scale bar = 20 μm. c) Morphology of Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical neurons in explant culture on rat glia . Cultures were sparsely labeled with GFP to visualize individual cells and their processes, and stained for GAD67 to exclude GABAergic neurons. Dendritic traces of GFP + neurons. Scale bar = 50 μm. d) Sholl analysis of Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical neurons in explant cultures shown in c). Shown is the number of crossings, dendritic length, terminal points, branch points and spines per 10 μm. Three independent experiments, 32 Rad21 lox/lox Nex Cre and 28 Rad21 +/+ Nex Cre neurons except for the number of spines (two independent experiments, 10 Rad21 lox/lox Nex Cre and 10 Rad21 +/+ Nex Cre neurons). * adj. P <0.05, ** adj. P <0.01, *** adj. P <0.001, **** adj. P <0.0001. Scale bar = 10 μm.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Immunofluorescence, Staining, Marker, Labeling

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) GSEA of the gene set downregulated (DEseq2, adj. P < 0.05) in RAD21-TEV neurons in Rad21 lox/lox Nex Cre neurons (left) and GSEA of genes downregulated in Rad21 lox/lox Nex Cre neurons (DEseq2, adj. P < 0.05) in RAD21-TEV neurons. b) Scatter plots of gene expression within aggregate GO terms, comparing RAD21-TEV with Rad21 lox/lox Nex Cre neurons. Genes that were found deregulated in at least one of the genotypes are shown. P -values and odds ratios refer to the probability of finding the observed patterns of co-regulation by chance. R S : Spearman’s rank coefficient. c) Deregulation of constitutive and activity-dependent genes 24h after acute cohesin depletion by inducible proteolytic cleavage of RAD21-TEV; adj. P <0.05 based on DEseq2 analysis of 3 RNA-seq replicates per experiment. Two independent experiments are shown.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Expressing, Activity Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Preferential deregulation in Rad21 lox/lox Nex Cre neurons of genes near constitutive and KCl-inducible neuronal enhancers . Based on 3 RiboTag RNA-seq replicates per genotype. b) Enrichment of inducible activity-dependent genes near constitutive and KCl-inducible neuronal enhancers . c) CTCF binding at neuronal genes and enhancers. All genes: all expressed genes in total RNA-seq; Activity-dependent genes: Previously defined activity-dependent genes (Kim et al., 2010) that are inducible by KCl in our experiments (KCl minus TTX adj. P < 0.05); Constitutive genes: Expressed genes that are not inducible by KCl in our experiments (KCl minus TTX adj. P > 0.05); Enhancers: Previously defined forebrain enhancers ; CTCF binding: Previously defined CTCF binding peaks within 1kb of TSS or enhancer. d) Only a minority of activity-dependent gene promoters are directly bound by CTCF, and most activity-dependent genes that lack CTCF promoter binding are nevertheless deregulated in cohesin-deficient neurons. e) Models of gene regulation by direct (left) versus domain-wide chromatin contacts (right). CD: contact domain.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: RNA Sequencing Assay, Activity Assay, Binding Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Top: The percentage of constitutive and activity-dependent genes deregulated in Rad21 lox/lox Nex Cre neurons in explant culture at baseline as determined by RNA-seq. Analysis of previously defined activity-dependent genes . Middle: Fraction of constitutive and activity-dependent genes deregulated in Rad21 lox/lox Nex Cre neurons in the presence of TTX and D-AP5 (TTX). Bottom: Fraction of constitutive and activity-dependent genes deregulated in Rad21 lox/lox Nex Cre neurons after 6h stimulation with KCl. b) Expression of activity-dependent genes in explant cultures of Rad21 +/+ and Rad21 lox/lox Nex Cre neurons under baseline conditions that allow for cell-cell communication versus TTX/D-AP5 (TTX). Comparison by two-sample Kolmogorov-Smirnov test showed that Rad21 +/+ Nex Cre neurons showed stronger expression of activity-dependent genes than Rad21 lox/lox Nex Cre neurons ( P = 5.89e-11). c) Fraction of activity-dependent genes significantly induced by KCl in Rad21 lox/lox Nex Cre neurons at 1 and 6h. In wild-type neurons, 117 and 810 genes were induced ≥2-fold at 1 and 6h of KCl treatment, respectively. d) Fraction of activity-dependent genes that were significantly induced by BDNF at 30 and 120min in RAD21-TEV neurons 24h after RAD21 cleavage. In control RAD21-TEV neurons, 16 and 261 activity-dependent genes were induced ≥2-fold 30 and 120min after BDNF treatment, respectively. Most of these remained inducible 24h after RAD21-TEV cleavage. Dark orange: Strongly induced: log2 FC>1, adj. P <0.05; light orange: moderately induced (log2 FC > 0.5, adj. P <0.05); grey: weakly induced (adj. P <0.05); white: not induced (adj. P >0.05).

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Activity Assay, RNA Sequencing Assay, Expressing

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Expression of the activity-dependent Fos gene at baseline, after TTX/D-AP5 (TTX), and KCl-stimulation (left, mean log2-transformed counts from 3 biological replicates, * adj. P < 0.05). b) Enhancer transcripts in control and Rad21 lox/lox Nex Cre neurons were quantified based on normalized RNA-seq reads within 1kb of the eRNA transcription start site. An intergenic region on chr11 was used as a negative control (71.177.622-71.177.792). c) H3K27ac ChIP normalized to H3 in control and Rad21 lox/lox Nex Cre neurons at a control site, Fos enhancer 1 and Fos enhancer 2 after TTX/D-AP5 (TTX) or 1h KCl (KCl). d) Interaction score heatmaps of the 65 kb region immediately surrounding Fos obtained by 5C. Black frames highlight interactions between the Fos gene and upstream enhancers 1 and 2. Previously published CTCF-ChIP-seq is shown for orientation and H3K27ac ChIP-seq in inactive (TTX-treated) and activated neurons is shown to annotate enhancer regions . RNA-seq in TTX-treated and 1h KCl-activated control and Rad21 lox/lox Nex Cre neurons shows KCl-inducible transcription of Fos enhancers in wild-type and cohesin-deficient neurons. Two independent biological replicates are shown in . e) Quantification of 5C contacts between the Fos promoter and Fos enhancer 1 (top), the Fos promoter and Fos enhancer 2 (middle), and CTCF-marked boundaries of the sub-TAD containing Fos (bottom). Two replicates per genotype and condition. f) Model for how cohesin-mediated domain-wide contacts alter the probability of enhancer-promoter contacts, and in this way fine-tune the transcription of activity-dependent genes at baseline and in response to activation. In the absence of cohesin, many activity-dependent genes are expressed at inappropriate levels, but most remain responsive to inducing activation signals. At the Fos and Arc loci, cohesin is not required for chromatin contacts between inducible enhancers and their target promoters. See text for details.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Expressing, Activity Assay, Transformation Assay, RNA Sequencing Assay, Negative Control, ChIP-sequencing, Activation Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Expression of Arc mRNA at baseline, after TTX/D-AP5 (TTX), and KCl-stimulation (top, mean log2-transformed counts from 3 biological RNA-seq replicates, * adj. P < 0.05) and at the indicated time after BDNF stimulation (bottom, data points represent biological RT-PCR replicates). P- values refer to induction relative to 0 min. * P < 0.05, *** P <0.001. b) Interaction score heatmaps of the ∼40 kb region immediately surrounding Arc obtained by 5C for resting (TTX) and 1h KCl-activated wild-type (top) and Rad21 lox/lox Nex Cre neurons (bottom). Black frames highlight interaction between the Arc gene (y-axis) and a nearby downstream enhancer (x-axis). Previously published CTCF-ChIP-seq is shown . H3K27ac ChIP-seq in inactive (TTX-treated) and activated neurons is shown to annotate enhancer regions . Two independent biological replicates are shown in . c) Quantification of 5C data. Arc enhancer-promoter loop (top). A CTCF-based loop that braces the Arc locus (arrowhead marked with * in panel b) is quantified for comparison (bottom).

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Expressing, Transformation Assay, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, ChIP-sequencing

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Top: Interaction frequency zoom-in heatmaps of 250 kb region surrounding the Fos gene. Dashed lines and arrow heads mark major CTCF binding sites at the boundaries of the domain that contains Fos . Note the weakening of these contacts in Rad21 lox/lox Nex Cre neurons. Bottom: Interaction score heatmaps of the 65 kb region immediately surrounding the Fos gene. Black frames highlight interactions between the Fos gene and upstream enhancers 1 and 2. H3K27ac ChIP-seq data from Bicuculline-treated (active) and TTX-treated (inactive) neurons annotate enhancer regions. Two independent biological replicates are shown. b) Top: Interaction frequency zoom-in heatmaps of ∼200 kb region surrounding the Arc gene. Dashed lines and arrow heads mark major CTCF binding sites at the boundaries of domains that contain the Arc locus. Note the weakening of these contacts in Rad21 lox/lox Nex Cre neurons. Bottom: Interaction score heatmaps of the ∼40 kb region immediately surrounding the Arc gene. Black frames highlight interaction between the Arc gene (y-axis) and a nearby downstream enhancer (x-axis). H3K27ac ChIP-seq data from Bicuculline-treated (active) and TTX-treated (inactive) neurons annotate enhancer regions. Two independent biological replicates are shown.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Binding Assay, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: ARID1A regulates DNA repair through chromatin organization and its deficiency triggers DNA damage-mediated anti-tumor immune response

doi: 10.1093/nar/gkae233

Figure Lengend Snippet: ARID1A is required for efficient chromatin loop formation. ( A – C ) 4C–seq normalized read counts at indicated viewpoints as well as differential 4C–seq track (log 2 +DSB/–DSB) in WT (grey) ARID1A-KO (blue) cells collected at indicated time points (T0 and T4). 4C–seq data were smoothed using 10-kb spans. ( D – F ) Box plots showing the differential 4C–seq (log 2 +DSB/–DSB) at indicated viewpoints. Four technical replicates from two independent experiments (biological replicates), data are presented mean ± SD, Student's t test. ( G – I ) Enrichment of γH2AX, RAD21 and CTCF, respectively, at the indicated DSBs in WT and ARID1A-KO cells, measured by ChIP-qPCR. n = 3 independent experiments (biological replicates); data are presented as mean ± SEM, Student's t test. Statistical significance is presented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant.

Article Snippet: Rabbit-anti-RAD21 (Abcam, Cat#ab992).

Techniques:

Journal: Chemical Science

Article Title: Discovery of a small molecule targeting ULK1-modulated cell death of triple negative breast cancer in vitro and in vivo † †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc05368h Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.

doi: 10.1039/c6sc05368h

Figure Lengend Snippet: Cell-specific microarray-based analysis of core ULK1-regulated network in MDA-MB-231 cells. (A) Expression data for microarray analysis from two individual experiments. MDA-MB-231 cells were treated with ULK1 siRNA or transfected with pcDNA 3.1-ULK1. Significant differentially expressed genes with opposite expression change are circled by red dashed lines. (B) 35 opposite expression changed genes. (C) Microarray-based ULK1 subnetwork in breast cancer, 23 opposite expression changed genes (blue) were involved in this subnetwork. ATF3, RAD21 and caspase3 were verified to be regulated by ULK1.

Article Snippet: Antibodies used in this study were as follows: ULK1 (8054, CST, MA, USA), ULK1 (ab128859, Abcam, UK), p-ULK1 ser317 (12753, CST), p-ULK1 ser757 (14202, CST), mAtg13 (13273, CST), p-mATG13 ser318 (PAB19948, Abnova, Taiwan), ATG101 (13492, CST), FIP200 (12436, CST), AMPKα (5831, CST), p-AMPKα thr172 (2535, CST), ACC (3676, CST), p-ACC ser79 (11818, CST), Beclin-1 (3495, CST), LC3B (3868, CST), p62 (5114, CST), ATG5 (12994, CST), PARP (9532, CST), caspase-3 (9665, CST), ATF3 (ab58668, Abcam), RAD21 (12673, CST), β-actin (66009-1-Ig, Proteintech, IL, USA).

Techniques: Microarray, Expressing, Transfection

Journal: Chemical Science

Article Title: Discovery of a small molecule targeting ULK1-modulated cell death of triple negative breast cancer in vitro and in vivo † †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc05368h Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.

doi: 10.1039/c6sc05368h

Figure Lengend Snippet: LYN-1604 induces autophagy involved in ATF3, RAD21, and caspase3. (A) After treatment with LYN-1604, MDA-MB-231 cells were stained with Hoechst 33258. Morphological changes were observed under fluorescence microscope. Scale bar = 20 μm. (B) After LYN-1604 treatment, the expression levels of caspase3 and PARP were determined by western blot analysis. β-Actin was measured as loading control. (C) After LYN-1604 treatment, the expression levels of ATF3 and RAD21 were determined by western blot analysis. β-Actin was measured as loading control. (D) MDA-MB-231 cells were transfected with control or ULK1 siRNA, followed by treatment with 2.0 μM LYN-1604 for 24 h. Then, the expression levels of cleaved-caspase3, ATF3 and RAD21 were determined by western blot analysis. β-Actin was measured as loading control. (E) The schematic model of LYN-1604-induced cell death pathways, associated with autophagy and apoptosis.

Article Snippet: Antibodies used in this study were as follows: ULK1 (8054, CST, MA, USA), ULK1 (ab128859, Abcam, UK), p-ULK1 ser317 (12753, CST), p-ULK1 ser757 (14202, CST), mAtg13 (13273, CST), p-mATG13 ser318 (PAB19948, Abnova, Taiwan), ATG101 (13492, CST), FIP200 (12436, CST), AMPKα (5831, CST), p-AMPKα thr172 (2535, CST), ACC (3676, CST), p-ACC ser79 (11818, CST), Beclin-1 (3495, CST), LC3B (3868, CST), p62 (5114, CST), ATG5 (12994, CST), PARP (9532, CST), caspase-3 (9665, CST), ATF3 (ab58668, Abcam), RAD21 (12673, CST), β-actin (66009-1-Ig, Proteintech, IL, USA).

Techniques: Staining, Fluorescence, Microscopy, Expressing, Western Blot, Control, Transfection