Journal: Molecular Cell

Article Title: Decreased Enhancer-Promoter Proximity Accompanying Enhancer Activation

doi: 10.1016/j.molcel.2019.07.038

Figure Lengend Snippet: Loss of Shh-Brain-Enhancer Proximity during Neuronal Differentiation (A) Map of the Shh regulatory domain showing the genes (black boxes), enhancers (green bars), and fosmid FISH probes (gray boxes). Probe and enhancer coordinates are listed in . (B) Violin plots showing the distribution of inter-probe distances (μm) between Shh and SBE6, SBE4, SBE2/3, ZRS, and CTRL probes in the nuclei of ESCs and D7 NPCs. Distances below the dotted horizontal line at 0.2 μm are considered co-localized. The asterisks on the FISH data represent Mann-Whitney U test significance between ESC and NPC populations. ∗∗ p < 0.01. Each violin plot represents one biological replicate; for other replicates and statistics, see Figure S1 and . (C) 3D-SIM images illustrating Shh -SBE6 separation in ESCs or in D7 NPCs. Scales bars are 5 μm (top two rows) and 1 μm (bottom, inset from center row). (D) Shh , Oct4 , and Nestin expression assayed by qRT-PCR during a time course of NPC differentiation. The graph shows mean (±SEM) log2 mRNA levels relative to Gapdh and normalized to the level in ESC (three technical replicates). (E) Violin plots showing Shh -SBE6 inter-probe distances in cell populations corresponding to the expression data in (D). (F) Kernel density plots showing Shh mRNA expression in single NPCs relative to Gapdh and normalized to the expression in ESC. Density is an arbitrary unit based on the frequency of the occurrence and the total counts and the size of the population (i.e., the binning of the data). Data from a biological replicate are shown in Figure S1 B.

Article Snippet: Each well of a 96-well PCR plate was loaded with 5 μl 2x Reaction Mix, 0.2 μl Superscript III RT/Platinum Taq Mix with RNaseOUT Ribonuclease Inhibitor (Invitrogen Cells Direct One-Step qRT-PCR kit, Life Technologies), 2.5 μl primer mix (containing 200 nM of each gene-specific primer), 1.3 μl H 2 O. Single-cell suspensions were sorted on their GFP reporter into separate wells of the 96-well PCR plate.

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

Journal: Molecular Cell

Article Title: Decreased Enhancer-Promoter Proximity Accompanying Enhancer Activation

doi: 10.1016/j.molcel.2019.07.038

Figure Lengend Snippet: Synthetic Activation of Shh and Increased Enhancer-Promoter Separation Using TALE-VP16 (A) Schematic of TALE-VP64 and TALE-VP128 constructs targeting the Shh promoter (tShh), SBE6, or SBE2. Repeat variable diresidue (RVD) code is displayed with one-letter abbreviations for amino acids. Self-cleaving (2A) peptide allows the expression of eGFP and cell isolation by fluorescence-activated cell sorting (FACS). A map of the targeting sites is shown at right. (B) Log2 mRNA levels of Shh, relative to Gapdh, assayed by qRT-PCR after TALE-VP64/128 expression in ESCs. Data show means (±SEMs) of three biological replicates normalized to ESCs expressing a control eGFP. (C) Violin plots representing one biological replicate of Shh-SBE6 inter-probe distances (μm) in ESCs expressing control eGFP, TALE-VP128 fusions targeting Shh promoter (tShh), SBE6, or SBE2. ∗∗ p < 0.01. Statistical data and replicate experiments are in . Distances below the dotted horizontal line at 0.2 μm are considered co-localized. (D) As in (C), but for VP64 recruitment to both SBE6 and SBE2 simultaneously (tSBE6+2), or a TALE with no fusion protein (tSBE6+2)-Δ. ∗∗ p < 0.01. Representative FISH images with probes for Shh (green) and SBE6 (red) in mESCs expressing tSBE(6+2)-VP64 and tSBE(6+2)-Δ are shown at right. (E) 5C heatmaps of the Shh regulatory region (chr5:28604000-29780000) with 16 kb binning and smoothing for ESCs and for ESCs expressing TALE-VP64 fusions targeting both SBE6 and SBE2. Difference 5C plots are shown above the main 5C plots. (F) Schematic representing TALE-LDB1 targeting sequences. (G) Three-color FISH with probes for Shh (green), SBE6 (magenta), and SBE2 (red) in mESCs expressing tShh-LDB1+tSBE2-LDB1. (H) Violin plots displaying Shh and SBE6 inter-probe distances (μm) in ESCs expressing eGFP or tShh-LDB1+tSBE6-LDB1. ∗∗ p < 0.01. (I) As in (H), but in cells expressing tShh-LDB1+tSBE2-LDB1. Shh-SBE6 distances are shown in the left-hand panel, and Shh-SBE2 distances are in the right-hand panel. ∗∗ p < 0.01. Statistical data relating to this figure are included in .

Article Snippet: Each well of a 96-well PCR plate was loaded with 5 μl 2x Reaction Mix, 0.2 μl Superscript III RT/Platinum Taq Mix with RNaseOUT Ribonuclease Inhibitor (Invitrogen Cells Direct One-Step qRT-PCR kit, Life Technologies), 2.5 μl primer mix (containing 200 nM of each gene-specific primer), 1.3 μl H 2 O. Single-cell suspensions were sorted on their GFP reporter into separate wells of the 96-well PCR plate.

Techniques: Activation Assay, Construct, Expressing, Cell Isolation, Fluorescence, FACS, Quantitative RT-PCR

Journal: Molecular Cell

Article Title: Decreased Enhancer-Promoter Proximity Accompanying Enhancer Activation

doi: 10.1016/j.molcel.2019.07.038

Figure Lengend Snippet:

Article Snippet: Each well of a 96-well PCR plate was loaded with 5 μl 2x Reaction Mix, 0.2 μl Superscript III RT/Platinum Taq Mix with RNaseOUT Ribonuclease Inhibitor (Invitrogen Cells Direct One-Step qRT-PCR kit, Life Technologies), 2.5 μl primer mix (containing 200 nM of each gene-specific primer), 1.3 μl H 2 O. Single-cell suspensions were sorted on their GFP reporter into separate wells of the 96-well PCR plate.

Techniques: Modification, Recombinant, Protease Inhibitor, Transfection, SYBR Green Assay, Staining, Whole Genome Amplification, Purification, Sequencing, Derivative Assay, Multiplex Assay, Software, Imaging, Microarray, Transformation Assay, RNA Sequencing Assay

Journal: Molecular Cell

Article Title: Decreased Enhancer-Promoter Proximity Accompanying Enhancer Activation

doi: 10.1016/j.molcel.2019.07.038

Figure Lengend Snippet:

Article Snippet: Mouse Cot-1 DNA , Invitrogen, ThermoFisher , 18440-016.

Techniques: Virus, Modification, Recombinant, Protease Inhibitor, Transfection, SYBR Green Assay, Staining, Reverse Transcription, Whole Genome Amplification, Purification, Sequencing, Derivative Assay, Multiplex Assay, Software, Imaging, Microarray, Transformation Assay, RNA Sequencing

Journal: Cell Metabolism

Article Title: Adipo-glial signaling mediates metabolic adaptation in peripheral nerve regeneration

doi: 10.1016/j.cmet.2023.10.017

Figure Lengend Snippet:

Article Snippet: Primary antibodies: Dendra2 (1:500, polyclonal rabbit, cat.#:ABIN361314, antibodies-online.com , RRID: AB_10789591 ), Wheat Germ Agglutinin-Alexa Fluor 488 Conjugate (WGA, 1:200, cat.#: W11261, Invitrogen), Leptin/Obese Receptor (OBRb) IgG (1:100, monoclonal mouse, cat.#: OBR12-A, Bio Trend), S100B (EP1576Y clone, 1:200, polyclonal rabbit, cat.#: ab52642, Abcam), MBP (1:2000, polyclonal chicken, cat.#: PA1-10008, Invitrogen, RRID: AB_1077024 ), IBA1 (1:400, polyclonal rabbit, cat.#: 019-19741, Wako, RRID: AB_839504 ), SOX10 (EPR4007-104, 1:100, monoclonal rabbit, cat.#: ab180862, Abcam, RRID: AB_2721184 ), PCNA (1:1000, monoclonal mouse, cat.#: ab29, Abcam; RRID: AB_303394 ), Total OXPHOS Rodent WB Antibody Cocktail (1:1000, monoclonal mouse, cat.#: ab110413, Abcam, RRID: AB_2629281 ), Neurofilament H (1:1000, polyclonal chicken, cat# 822601, Bioloegend, RRID: AB_2564859 ), PDGFRa (1:100, polyclonal goat, cat#1062, R&D systems, RRID: AB_2236897 Secondary antibodies: Cy3-goat-anti-rabbit (1:1000, cat.#:111-165-144, Jackson Immuno, RRID: AB_2338006 ), Cy3-goat-anti-mouse (1:1000, cat.#:115-165-071, Jackson Immuno, RRID: AB_2338687 ), Alexa Fluor Plus 488 Goat anti-Chicken IgY (H+L) Cross-Adsorbed (1:1000, cat.#: A32931, Invitrogen), Alexa Fluor 488 Goat Anti-Rabbit (1:1000, cat.#: 111-545-144, Jackson Immuno, RRID: AB_2762843 ).

Techniques: Recombinant, Electron Microscopy, Protease Inhibitor, Picogreen Assay, Microarray, RNA Sequencing, Real-time Polymerase Chain Reaction, Software

Journal: World Journal of Gastrointestinal Surgery

Article Title: Research progress on O-GlcNAcylation in the occurrence, development, and treatment of colorectal cancer

doi: 10.4240/wjgs.v13.i2.96

Figure Lengend Snippet: Summary of Studies on O-GlcNAcylation and colorectal cancer in recent 5 years

Article Snippet: Harosh-Davidovich et al [ ] , 2018 , CT26 murine colon carcinoma cells and NIH-3T3 murine fibroblasts , ATCC , (1) Protein extraction. β-Catenin IP. (2) Affinity purification of β-catenin with Wheat Germ Agglutinin (WGA). (3) WB. (4) Cell motility assay. (5) OGA and OGT silencing. (6) Luciferase reporter assays. (7) qRT-PCR. And (8) In vivo orthotopic mouse model of CRC , O-GlcNAcylation may enhance the proliferation and metastasis of CRC by regulating the expression of catenin and E-cadherin, which proves the influence of O-GlcNAcylation on the poor prognosis of CRC patients.

Techniques: Glycoproteomics, Quantitative Proteomics, Plasmid Preparation, Transfection, Isolation, Purification, RNA Extraction, Protein Extraction, Western Blot, Flow Cytometry, Biomarker Discovery, Expressing, Migration, Immunofluorescence, Confocal Microscopy, Invasion Assay, Co-culture Assay, Gene Expression, Chromatin Immunoprecipitation, Construct, CRISPR, Knock-Out, Knockdown, shRNA, Marker, Activation Assay, Labeling, Microarray, Immunohistochemistry-IF, Activity Assay, Soft Agar Assay, Ligation, In Vitro, Cell Culture, Stable Transfection, CCK-8 Assay, Virus, Reporter Assay, Reverse Transcription, Viability Assay, Colony Assay, Cell Migration Assay, Infection, Microscopy, Mutagenesis, Immunohistochemical staining, Staining, Sequencing, DNA Methylation Assay, Mass Spectrometry, Multiplex Assay, Immunohistochemistry, Proteomic Assay, Affinity Purification, Motility Assay, Luciferase, In Vivo, Fluorescence, In Situ Hybridization, Sample Prep, Concentration Assay, Chemotaxis Assay, Extraction, Bacteria, Adjuvant, Histopathology, Cell Adhesion Assay, Colorimetric Assay

Journal: World Journal of Gastrointestinal Surgery

Article Title: Research progress on O-GlcNAcylation in the occurrence, development, and treatment of colorectal cancer

doi: 10.4240/wjgs.v13.i2.96

Figure Lengend Snippet: Summary of Studies on O-GlcNAcylation and colorectal cancer in recent 5 years

Article Snippet: Harosh-Davidovich et al [ ] , 2018 , CT26 murine colon carcinoma cells and NIH-3T3 murine fibroblasts , ATCC , (1) Protein extraction. β-Catenin IP. (2) Affinity purification of β-catenin with Wheat Germ Agglutinin (WGA). (3) WB. (4) Cell motility assay. (5) OGA and OGT silencing. (6) Luciferase reporter assays. (7) qRT-PCR. And (8) In vivo orthotopic mouse model of CRC , O-GlcNAcylation may enhance the proliferation and metastasis of CRC by regulating the expression of catenin and E-cadherin, which proves the influence of O-GlcNAcylation on the poor prognosis of CRC patients.

Techniques: Glycoproteomics, Quantitative Proteomics, Plasmid Preparation, Transfection, Isolation, Purification, RNA Extraction, Protein Extraction, Western Blot, Flow Cytometry, Biomarker Discovery, Expressing, Migration, Immunofluorescence, Confocal Microscopy, Invasion Assay, Co-culture Assay, Gene Expression, Chromatin Immunoprecipitation, Construct, CRISPR, Knock-Out, Knockdown, shRNA, Marker, Activation Assay, Labeling, Microarray, Immunohistochemistry-IF, Activity Assay, Soft Agar Assay, Ligation, In Vitro, Cell Culture, Stable Transfection, CCK-8 Assay, Virus, Reporter Assay, Reverse Transcription, Viability Assay, Colony Assay, Cell Migration Assay, Infection, Microscopy, Mutagenesis, Immunohistochemical staining, Staining, Sequencing, DNA Methylation Assay, Mass Spectrometry, Multiplex Assay, Immunohistochemistry, Proteomic Assay, Affinity Purification, Motility Assay, Luciferase, In Vivo, Fluorescence, In Situ Hybridization, Sample Prep, Concentration Assay, Chemotaxis Assay, Extraction, Bacteria, Adjuvant, Histopathology, Cell Adhesion Assay, Colorimetric Assay