Journal: Cells

Article Title: NeuroHeal Improves Muscle Regeneration after Injury

doi: 10.3390/cells10010022

Figure Lengend Snippet: NeuroHeal modulates differentiation process and increases myoblast fusion in vitro. ( A ) Left, representative microphotographs of differentiated C2C12 myoblast cell line immunostained for Ki67, Pax7, and MyoD and stained with DAPI from the different experimental groups at 3 days of differentiation: atrophy-induced treated with vehicle (Veh), atrophy-induced treated with Ex-527 (Ex-527), atrophy-induced treated with NeuroHeal (NH), and atrophy-induced treated with NH plus Ex-527. Scale bar 25 μm and identical for all corresponding microphotographs as represented in the first image panel, the vehicle condition. Right, bar graph of the average number of positive nuclei for Ki67 and Pax7, and the relative intensity per cell of MyoD ( n = 3; one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001). ( B ) Left, representative immunofluorescence microphotographs of differentiated C2C12 myoblast cell line immunostained for MyHC and stained with DAPI (blue) from the different experimental groups at 5 days of differentiation. Panels a and b are zoom images for each condition from the images of the left. The scale bar is 200 μm for all corresponding microphotographs as represented in the first image panel, the vehicle condition, and zoomed images. Right, bar graph of the percentage of the number of positive myotubes with three or more nuclei ( n = 3, one-way ANOVA, * p < 0.05).

Article Snippet: The antibodies used were rabbit anti-ki67 (1:100; Abcam), mouse anti-MyoD (1:150; Santa Cruz Biotechnology), mouse anti-Myosin heavy chain (all fast isoforms) (MyHC, 1:20, DSHB), rabbit anti-parvalbumin (1:100, Swant), and mouse anti-Pax7 (1:100; DSHB).

Techniques: In Vitro, Staining, Immunofluorescence

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: International Journal of Molecular Sciences

Article Title: Scratching Counteracts IL-13 Signaling by Upregulating the Decoy Receptor IL-13Rα2 in Keratinocytes

doi: 10.3390/ijms20133324

Figure Lengend Snippet: Immunofluorescence analysis for IL-13Rα2 ( A ) and IL-13Rα1 ( B ) proteins. Non-scratched control or scratched confluent keratinocyte sheets were immunostained with anti-IL-13Rα2 or anti-IL-13Rα1 antibodies. Data is shown as the mean ± SEM. * p < 0.05. Scale bar: 50 µm.

Article Snippet: The antibodies for immunofluorescence and immunohistochemistry staining were used as follows: Anti-IL-13Rα2 mouse monoclonal antibody (Abcam, Cambridge, UK), normal mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA), and goat anti-mouse IgG conjugated with Alexa Fluor 488 dye (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Scratching Counteracts IL-13 Signaling by Upregulating the Decoy Receptor IL-13Rα2 in Keratinocytes

doi: 10.3390/ijms20133324

Figure Lengend Snippet: Immunohistochemical analysis for IL-13Rα2 expression. Control normal skin and lichenified lesional AD skin were immunostained with control IgG and anti-IL-13Rα2 antibody ( A ). The percentage of IL-13Rα2 positive keratinocytes was calculated in 11 normal skin and 11 AD skin samples ( B ). Data is shown as the mean ± SEM. *** p < 0.001. Scale bar: 50 µm.

Article Snippet: The antibodies for immunofluorescence and immunohistochemistry staining were used as follows: Anti-IL-13Rα2 mouse monoclonal antibody (Abcam, Cambridge, UK), normal mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA), and goat anti-mouse IgG conjugated with Alexa Fluor 488 dye (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Immunohistochemical staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Scratching Counteracts IL-13 Signaling by Upregulating the Decoy Receptor IL-13Rα2 in Keratinocytes

doi: 10.3390/ijms20133324

Figure Lengend Snippet: IL13RA2 expression was upregulated in the IL-13Rα2-Tg-HaCaT cells more than in control Moc-HaCaT cells ( A ). Upregulated IL-13Rα2 protein expression was observed in the IL-13Rα2-Tg-HaCaT cells, compared to that in Moc-HaCaT cells ( B ). IL-13-induced IVL downregulation was partially restored in IL-13Rα2-Tg-HaCaT cells ( C ). ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies for immunofluorescence and immunohistochemistry staining were used as follows: Anti-IL-13Rα2 mouse monoclonal antibody (Abcam, Cambridge, UK), normal mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA), and goat anti-mouse IgG conjugated with Alexa Fluor 488 dye (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing

Journal: European Respiratory Review

Article Title: Selection of potential targets for stratifying congenital pulmonary airway malformation patients with molecular imaging: is MUC1 the one?

doi: 10.1183/16000617.0217-2023

Figure Lengend Snippet: Expression of targets described as upregulated in both congenital pulmonary airway malformations (CPAM) and adenocarcinoma in situ (AIS)

Article Snippet: Primary antibodies used for immunofluorescent staining were MUC1 (AMAb191533, mouse, 1:100; Atlas antibodies), SOX2 (14-9811-82, rat, 1:800; ThermoFisher), CDH1 (sc-8426, mouse, 1:500; Santa Cruz), ERBB2 (4290, rabbit 1:250; Cell signalling), CEACAM5 (7072, mouse, 1:50; Dako).

Techniques: Expressing, In Situ, Membrane

Journal: European Respiratory Review

Article Title: Selection of potential targets for stratifying congenital pulmonary airway malformation patients with molecular imaging: is MUC1 the one?

doi: 10.1183/16000617.0217-2023

Figure Lengend Snippet: a) Haematoxylin and eosin (H&E) staining of adjacent “normal” lung tissue, congenital pulmonary airway malformation (CPAM) tissue without KRAS mutations (KRAS − ), CPAM tissue with KRAS mutations (KRAS + ) and areas with mucinous proliferations (MP) found in only KRAS + CPAM tissue. Scale bars=100 µm. b) Immunofluorescent staining of KRAS − CPAM tissue, KRAS + CPAM tissue and MP within KRAS + CPAM tissue with mucin 1 (MUC1), sex-determining region Y-box 2 (SOX2) and 4′,6-diamidino-2-phenylindole (DAPI). Scale bars=100 µm (top row), 50 µm (bottom rows). c) H&E staining of MPs, and immunofluorescent staining of MUC1, SOX2 and DAPI. Scale bars=50 µm. Arrows point to positive signals.

Article Snippet: Primary antibodies used for immunofluorescent staining were MUC1 (AMAb191533, mouse, 1:100; Atlas antibodies), SOX2 (14-9811-82, rat, 1:800; ThermoFisher), CDH1 (sc-8426, mouse, 1:500; Santa Cruz), ERBB2 (4290, rabbit 1:250; Cell signalling), CEACAM5 (7072, mouse, 1:50; Dako).

Techniques: Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Cellular Tight Junctions Prevent Effective Campylobacter jejuni Invasion and Inflammatory Barrier Disruption Promoting Bacterial Invasion from Lateral Membrane in Polarized Intestinal Epithelial Cells

doi: 10.3389/fcimb.2018.00015

Figure Lengend Snippet: TJ disruption is tightly associated with lipid raft-mediated C. jejuni invasion in polarized epithelial cells. (A–F) Caco-2 cells cultured for 7 days on transwells and treated with 4 mM EGTA for 20 min. Following EGTA treatment, cells were incubated in normal culture medium. During the incubation, the TER value (A) and ZO-1 localization (B–F) were evaluated every hour for 3 h. TER values were calculated as the percentage of the TER value for untreated cells. Scale bar = 10 μm. (G) Caco-2 cells treated with 4 mM EGTA for 20 min were incubated in normal culture medium for the indicated time. After incubation, cells were pretreated with 10 mM MβCD for 1 h and infected with C. jejuni for 6 h. The number of intracellular bacteria was measured using a gentamycin protection assay. Results are shown as the mean ± SD; n = 4. Significant difference from the post Ca 2+ reintroduction 0 h group are shown: N.S.; not significant; * P < 0.05; ** P < 0.01.

Article Snippet: Antibodies to the following were diluted in 3% BSA and used for Immunofluorescence staining ZO-1 (1:200; BD Bioscience), Alexa Fluor 568 (1:200, Molecular Probes).

Techniques: Cell Culture, Incubation, Infection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Cellular Tight Junctions Prevent Effective Campylobacter jejuni Invasion and Inflammatory Barrier Disruption Promoting Bacterial Invasion from Lateral Membrane in Polarized Intestinal Epithelial Cells

doi: 10.3389/fcimb.2018.00015

Figure Lengend Snippet: C. jejuni invasion is increased by active inflammation in polarized epithelial cells. (A–C) Caco-2 cells were treated with 20 ng/ml TNF-α for 48 h. Following TNF-α treatment, TER values (A) and ZO-1 localization (B,C) were evaluated. Scale bar = 10 μm. (D) Caco-2 cells were treated with 20 ng/ml TNF-α followed by treatment with 10 mM MβCD for 1 h and were infected with C. jejuni for 6 h. The number of intracellular bacteria was measured by gentamycin protection assay. Results are shown as the mean ± SD n = 4. Significant difference from the control or EGTA treated group are shown: N.S.; not significant; ** P < 0.01.

Article Snippet: Antibodies to the following were diluted in 3% BSA and used for Immunofluorescence staining ZO-1 (1:200; BD Bioscience), Alexa Fluor 568 (1:200, Molecular Probes).

Techniques: Infection

Journal: Frontiers in Microbiology

Article Title: β-Actin: Not a Suitable Internal Control of Hepatic Fibrosis Caused by Schistosoma japonicum

doi: 10.3389/fmicb.2019.00066

Figure Lengend Snippet: The expression profile of hepatic β-actin in S. japonicum infected mice. (A) Total liver lysates were subjected to detect the expression levels of β-actin, GAPDH, and β-Tubulin by western blot. (B) Densitometric analysis of β-actin, GAPDH, and β-Tubulin using Image J2x software. The fold changes were calculated by comparing to the control group. (C) Absolute quantification of β-actin by real-time quantitative PCR. Data represent the mean ± SE. ∗ P < 0.05, infected groups vs control group; ∗∗ P < 0.01, infected groups vs control group; ∗∗∗ P < 0.001, infected groups vs control group. && P < 0.01.

Article Snippet: Equal amounts (60 μg) of protein were loaded and separated on a 10% polyacrylamide gel with 200 V for 2 h and then transferred to a 0.22 μm PVDF membrane (Millipore, MA, United States) with 300 mA for 3 h. After blocking in PBS containing 5% milk and 0.1% Tween-20 for 2 h at room temperature, the membranes were incubated with primary antibodies mouse anti-β-actin (58169, Cell Signaling Technology, Boston, MA, United States, used at 1:2000 dilution), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich, St. Louis, MO, United States, used at 1:2000 dilution), and mouse anti-β-Tubulin (T0198, Sigma-Aldrich, St. Louis, MO, United States, used at 1:2000 dilution), rabbit anti-α-SMA (19245, Cell Signaling Technology, Boston, MA, United States, used at 1:1000 dilution), rabbit anti-Collagen I (COL1A1) (BA0325, Boster, Wuhan, China, used at 1:1000 dilution), rabbit anti-Collagen III (COL3A1) (BM1625, Boster, Wuhan, China, used at 1:1000 dilution) at 4°C overnight.

Techniques: Expressing, Infection, Western Blot, Software, Control, Quantitative Proteomics, Real-time Polymerase Chain Reaction

Journal: Frontiers in Microbiology

Article Title: β-Actin: Not a Suitable Internal Control of Hepatic Fibrosis Caused by Schistosoma japonicum

doi: 10.3389/fmicb.2019.00066

Figure Lengend Snippet: Co-localization of β-actin and α-SMA in S. japonicum infected mice liver. (A) Co-immunofluorescence staining for β-actin (green) and α-SMA (red). (B) The integrated optical densities (IODs) of β-actin and α-SMA were analyzed by Image Pro Plus 6.0 software. The correlation between the IOD of β-actin and α-SMA was analyzed by Spearman’s correlation analysis. (C) The correlation between the concentration of hepatic hydroxyproline and the level of β-actin mRNA in mice was analyzed by Spearman’s correlation analysis.

Article Snippet: Equal amounts (60 μg) of protein were loaded and separated on a 10% polyacrylamide gel with 200 V for 2 h and then transferred to a 0.22 μm PVDF membrane (Millipore, MA, United States) with 300 mA for 3 h. After blocking in PBS containing 5% milk and 0.1% Tween-20 for 2 h at room temperature, the membranes were incubated with primary antibodies mouse anti-β-actin (58169, Cell Signaling Technology, Boston, MA, United States, used at 1:2000 dilution), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich, St. Louis, MO, United States, used at 1:2000 dilution), and mouse anti-β-Tubulin (T0198, Sigma-Aldrich, St. Louis, MO, United States, used at 1:2000 dilution), rabbit anti-α-SMA (19245, Cell Signaling Technology, Boston, MA, United States, used at 1:1000 dilution), rabbit anti-Collagen I (COL1A1) (BA0325, Boster, Wuhan, China, used at 1:1000 dilution), rabbit anti-Collagen III (COL3A1) (BM1625, Boster, Wuhan, China, used at 1:1000 dilution) at 4°C overnight.

Techniques: Infection, Immunofluorescence, Staining, Software, Concentration Assay

Journal: Oncotarget

Article Title: CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma

doi:

Figure Lengend Snippet: (A) Both qRT-PCR and Western blotting assays verified that CtBP2 expression was increased after stable transfection of CtBP2 expression plasmid. (B) The migration capacity of Huh7 cells, as assessed by wound healing assays, was enhanced by CtBP2 overexpression. (C) Transwell invasion assays demonstrated that elevating CtBP2 expression increased the invasive capability of Huh7 cells. (D) Overexpression of CtBP2 decreased E-cadherin expression and increased the expression of N-cadherin, Vimentin and Fibronectin in Huh7 cells, as measured by Western blotting. (E) Double immunofluorescence staining confirmed that CtBP2 overexpression resulted in the downregulation of E-cadherin and the upregulation of N-cadherin in Huh7 cells.

Article Snippet: Cell lysates (50 μg) were prepared in RIPA buffer...The following antibodies were used: mouse monoclonal antibodies to E-cadherin (Santa Cruz, CA, USA), Vimentin (Santa Cruz), β-actin (Boster Biotechnology, Wuhan, China) and rabbit polyclonal antibodies against GLI1, SNAI1, N-cadherin and CtBP2 (Abcam, Cambridge, UK).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Stable Transfection, Plasmid Preparation, Migration, Over Expression, Double Immunofluorescence Staining

Journal: Oncotarget

Article Title: CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma

doi:

Figure Lengend Snippet: (A) Both qRT-PCR and Western blotting assays confirmed that siRNA transfection eliminated CtBP2 expression in MHCC97H cells. (B) Wound healing assays indicated that CtBP2 siRNA suppressed MHCC97H cell migration. (C) The invasive ability of MHCC97H cells was repressed by CtBP2 siRNA in Transwell invasion assays. (D) Western blotting assays demonstrated that CtBP2 suppression lead to E-cadherin upregulation and N-cadherin, Vimentin and Fibronectin downregulation in MHCC97H cells.

Article Snippet: Cell lysates (50 μg) were prepared in RIPA buffer...The following antibodies were used: mouse monoclonal antibodies to E-cadherin (Santa Cruz, CA, USA), Vimentin (Santa Cruz), β-actin (Boster Biotechnology, Wuhan, China) and rabbit polyclonal antibodies against GLI1, SNAI1, N-cadherin and CtBP2 (Abcam, Cambridge, UK).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Expressing, Migration

Journal: Oncotarget

Article Title: CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma

doi:

Figure Lengend Snippet: (A) Both qRT-PCR and Western blotting assays demonstrated that SNAI1 siRNA abated SNAI1 expression (left panel) in Huh7 cells and that CtBP2 expression was enhanced by CtBP2 expression plasmid transfection in Huh7 cells without SNAI1. (B) Western blotting showed that CtBP2 overexpression lead to E-cadherin downregulation and N-cadherin, Vimentin and Fibronectin upregulation in Huh7 cells with normal SNAI1 expression. However, silencing SNAI1 counteracted the regulatory effect of CtBP2 on E-cadherin, N-cadherin, Vimentin and Fibronectin. (C) Co-IP assays demonstrated direct CtBP2 and SNAI1 binding in Huh7 GLI1 cells. (D) Both qRT-PCR and Western blotting verified that siRNA abolished CtBP2 expression in Huh7 GLI1 cells. (E) Western blotting assays demonstrated that CtBP2 knockdown in Huh7 GLI1 cells lead to E-cadherin upregulation and N-cadherin, Vimentin and Fibronectin downregulation, while SNAI1 expression was not significantly affected. However, silencing CtBP2 did not affect the expression of E-cadherin, N-cadherin and Fibronectin. Neither the expression of Vimentin nor the expression of SNAI1 could be detected in Huh7 Vector cells transfected with CtBP2 siRNAs or Scr siRNAs.

Article Snippet: Cell lysates (50 μg) were prepared in RIPA buffer...The following antibodies were used: mouse monoclonal antibodies to E-cadherin (Santa Cruz, CA, USA), Vimentin (Santa Cruz), β-actin (Boster Biotechnology, Wuhan, China) and rabbit polyclonal antibodies against GLI1, SNAI1, N-cadherin and CtBP2 (Abcam, Cambridge, UK).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Plasmid Preparation, Transfection, Over Expression, Co-Immunoprecipitation Assay, Binding Assay, Knockdown

Journal: Oncotarget

Article Title: CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma

doi:

Figure Lengend Snippet: GLI1 upregulates both SNAI1 and CtBP2 simultaneously. SNAI1 binds the E-cadherin promoter after binding CtBP2, leading to the EMT phenotype of HCC cells.

Article Snippet: Cell lysates (50 μg) were prepared in RIPA buffer...The following antibodies were used: mouse monoclonal antibodies to E-cadherin (Santa Cruz, CA, USA), Vimentin (Santa Cruz), β-actin (Boster Biotechnology, Wuhan, China) and rabbit polyclonal antibodies against GLI1, SNAI1, N-cadherin and CtBP2 (Abcam, Cambridge, UK).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: a Schematic illustration of bis-DbTACs design, which is based on DbTACs. b Schematic illustration of three ligand covalent sites of bis-DbTACs equivalent to a DNA tetrahedral scaffold with three polyA domains. Au NPs (5, 10, and 15 nm) correspond to CRBN, CDK9, and CDK6 ligands, respectively. c Cartoon and representative TEM images of bis-DbTACs equivalents. Scale bars are 75 Å and 200 Å, respectively. d WB analysis of the selectively targeted degradation ability of bis-DbTACs at different concentrations and semiquantitative analysis of the grayscale. The error bars indicate the mean ± SD values; n = 3. e Immunofluorescence double-staining images of HepG2 cells treated with/without bis-DbTACs were recorded by confocal laser scanning microscopy. The cell nucleus was stained with DAPI. CDK6 and CDK9 proteins were labeled with anti-CDK6 and anti-CDK9 antibodies, respectively. Scale bars, 10 μm.

Article Snippet: The primary antibody used was mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:100), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:100).

Techniques: Immunofluorescence, Double Staining, Confocal Laser Scanning Microscopy, Staining, Labeling