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: Cancers

Article Title: Principles of Molecular Utility for CMS Classification in Colorectal Cancer Management

doi: 10.3390/cancers15102746

Figure Lengend Snippet: Available gene expression assays predictive of prognosis in early-stage CRC.

Article Snippet: ColoPrint colon cancer recurrence assay , Agendia, Inc. , 18-gene expression profile , Fresh tissue or fresh, frozen tissue , Agendia customized whole-genome high-density microarrays , Not FDA approved , [ , , ] .

Techniques: Gene Expression, Expressing, Formalin-fixed Paraffin-Embedded, Amplification, Microarray, Derivative Assay

Journal: BMC Plant Biology

Article Title: Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa

doi: 10.1186/1471-2229-7-59

Figure Lengend Snippet: Microarray expression support for PoptrIAA3 subgroup . Data are expressed as fold change from negative controls, which consisted of the 95 th percentile signal from presumably unexpressed transposable element target sequences. Error bars represent standard errors from two biological replicates. G43h, germinant 43 hours after imbibition; ApB, apical bud; AxB, axillary bud; YFB, young female bud; YMB, young male bud; F, female catkin, post-fertilization; LPI1, leaf plastochron index 1; LPI2, leaf plastochron index 2; LPI5, leaf plastochron index 5; PC, phloem plus cortex. Samples are further defined in Additional file .

Article Snippet: We used a whole genome microarray that we designed together with NimbleGen Systems, Inc.

Techniques: Microarray, Expressing

Journal: Frontiers in Immunology

Article Title: Resolvin D1 Reduces Lung Infection and Inflammation Activating Resolution in Cystic Fibrosis

doi: 10.3389/fimmu.2020.00581

Figure Lengend Snippet: RvD1 regulates select genes related to microbial clearance and inflammatory signaling in CF MΦ and epithelial cells. (A) Homozygous ΔF508 peripheral blood monocyte-derived MΦ (0.5–1 × 10 6 cells/plate) and CFBEC (2.5 × 10 6 cells/plate) were treated with RvD1 (10 nM) or vehicle (0.01% EtOH) for 15 min at 37°C and infected with RP73 P. aeruginosa (∼7.5 × 10 6 CFU/plate). Medium was removed after 3 h, and total RNA was used for microarray analysis. Shown here are heat map view (Cluster 3.0, TreeView, Stanford University Labs) and scatter plots of gene expression patterns (mean from five different donors). (B) IPA findings of biological functions associated with RvD1-regulated genes in MΦ and CFBEC. (C) Top RvD1-regulated genes and associated functions identified by IPA. Blue symbols, upregulated genes; yellow symbols, downregulated genes; blue dotted lines, expression leading to activation; yellow dotted lines, expression leading to inhibition; gray dotted lines, expression leading to unpredictable effect of function; blue boxes, activated functions; yellow boxes, inhibited functions.

Article Snippet: Total RNA extracted from CFBEC and MΦ cells was linearly amplified, labeled with Cy3/5, and hybridized on HOA_007 Human Whole Genome OneArray Microarray V7 (29,264 probes; Phalanx Biotech, San Diego, CA, United States) analyzed as in ref. ( ).

Techniques: Derivative Assay, Infection, Microarray, Gene Expression, Expressing, Activation Assay, Inhibition