Journal: Science Advances

Article Title: Condensed but liquid-like domain organization of active chromatin regions in living human cells

doi: 10.1126/sciadv.adf1488

Figure Lengend Snippet: ( A ) Model of Cy3-labeled DNA structure with a side and top view (provided by Y. Joti at Spring-8). ( B ) FACS profiles of HeLa cells synchronized at the G1-S boundary, using a double thymidine block, and released in fresh medium at the indicated time point after release. ( C ) HeLa cell DNA replication foci labeled with a pulse of Cy3-dCTP (second row) 1 hour (first column) and 5 hours (second column) after release from a double thymidine block. The nuclei were stained with DAPI (top row, blue). ( D ) A scheme of Cy3-dCTP–labeled DNA immuno-purification and analysis. Synchronized HeLa cells in early and mid S phase were pulse-labeled with Cy3-dCTP. DNA from each cell fraction was purified, fragmented, and immunoprecipitated with an anti-Cy3 antibody. The enriched DNA fractions are indexed, amplified, and sequenced. ( E ) Visualization of Cy3-dCTP–labeled regions on chromosome 10 using the Integrative Genomics Viewer (IGV) Browser. ( F ) Cy3-labeled genome regions are enriched with active histone marks and excluded from an inactive mark: view from chr6_5512490–8384778 (also see fig. S3, A and B). ( G ) Venn diagrams show overlapped size (in bp) between Cy3-labeled regions and contact domains (left) and Cy3-labeled regions and A-compartment (right).

Article Snippet: For each assay, 200 ng of an enhanced green fluorescent protein (EGFP) gene polymerase chain reaction (PCR) fragment amplified by the Cy3 PCR labeling kit (PP-301S-CY3; Jena Bioscience) and the linearized plasmid (pGEM-T Easy plasmid digested with EcoRI) were resuspended in PBS buffer containing 300 ng of anti-Cy3 antibody (sc-166894; Santa Cruz Biotechnology).

Techniques: Labeling, Blocking Assay, Staining, Purification, Immunoprecipitation, Amplification

Journal: Science Advances

Article Title: Condensed but liquid-like domain organization of active chromatin regions in living human cells

doi: 10.1126/sciadv.adf1488

Figure Lengend Snippet: ( A ) Schematic of replication foci labeling with fluorescent nucleotides at high concentrations (left) and single-nucleosome labeling with a low concentration of fluorescent nucleotides (right). The inset shows that single-nucleosome labeling is based on the incorporation of a single nucleotide conjugated with fluorescent dye. This cartoon is simplified and fluorescent labeling can also be in the linker DNA region. ( B ) Experimental scheme for labeling and imaging. ( C ) Image of single-nucleosome dots labeled with Cy3-dCTP in a live HeLa nucleus. The bar represents 1 μm. See also movies S1 and S2. ( D ) Oblique illumination microscopy system. Only a thin optical layer within the nucleus (red) was illuminated with a low background using a sheet light (green). The cartoon was reproduced and modified with permission from . ( E ) Representative single-step photobleaching of a Cy3-labeled nucleosome dot. The vertical axis represents the fluorescence intensity and the horizontal axis is the tracking time series. A.U., arbitrary units. ( F ) Three representative trajectories of single-nucleosomes. The bar represents 100 nm.

Article Snippet: For each assay, 200 ng of an enhanced green fluorescent protein (EGFP) gene polymerase chain reaction (PCR) fragment amplified by the Cy3 PCR labeling kit (PP-301S-CY3; Jena Bioscience) and the linearized plasmid (pGEM-T Easy plasmid digested with EcoRI) were resuspended in PBS buffer containing 300 ng of anti-Cy3 antibody (sc-166894; Santa Cruz Biotechnology).

Techniques: Labeling, Concentration Assay, Imaging, Microscopy, Modification, Fluorescence

Journal: Science Advances

Article Title: Condensed but liquid-like domain organization of active chromatin regions in living human cells

doi: 10.1126/sciadv.adf1488

Figure Lengend Snippet: ( A ) Displacement (movement) distributions of single-nucleosomes labeled with Cy3-dCTP at active chromatin (euchromatic) regions in live HeLa cells for 50 ms ( n = 27 cells). ( B ) MSD plots (±SD among cells) of Cy3-labeled nucleosomes (red, n = 27 cells) and H2B-Halo–labeled nucleosomes [blue, data were reproduced from ] in living HeLa cells from 0 to 3 s. The Kolmogorov-Smirnov test was used to determine P values. * P < 0.05 for Cy3-dCTP versus H2B-Halo ( P = 0.049). *** P < 0.0001 for Cy3 nucleosomes versus FA-fixed Cy3 nucleosomes ( P = 2.1 × 10 −13 ), H2B-Halo nucleosomes versus FA-fixed H2B-Halo nucleosomes ( P = 5.7 × 10 −6 ). Not significant (N.S.) for FA-Cy3 nucleosomes versus FA-H2B-Halo nucleosomes ( P = 0.37). R c values (estimated radius of constraint of the nucleosome motion) are also shown. R c values of Cy3 and H2B-Halo nucleosomes are also significantly different: P = 0.049 by Kolmogorov-Smirnov test. ( C ) Log-log plots of MSD data shown in (B). The indicated straight lines were fitted using the data from 0.05 to 0.5 s. The plots cannot be fitted linearly beyond this time range, suggesting the motion mode changes over 0.5 s. ( D ) Schematic for nucleosome motion angle distribution analysis . Schematics were reproduced from . ( E ) Angle distributions of Cy3 (left) and H2B-Halo nucleosomes (right). Moving angles of single nucleosomes are biased toward 180° and the AC is negative, indicating that they are often pulled back to their original positions. H2B-Halo data were reproduced from .

Article Snippet: For each assay, 200 ng of an enhanced green fluorescent protein (EGFP) gene polymerase chain reaction (PCR) fragment amplified by the Cy3 PCR labeling kit (PP-301S-CY3; Jena Bioscience) and the linearized plasmid (pGEM-T Easy plasmid digested with EcoRI) were resuspended in PBS buffer containing 300 ng of anti-Cy3 antibody (sc-166894; Santa Cruz Biotechnology).

Techniques: Labeling

Journal: Science Advances

Article Title: Condensed but liquid-like domain organization of active chromatin regions in living human cells

doi: 10.1126/sciadv.adf1488

Figure Lengend Snippet: (A) Diagram of dual-color visualization of single-nucleosome dynamics. (B) Two hypothetical models of higher-order chromatin structure, condensed organization (left), or extended loops (right). In both models, cohesin holds the chromatin fiber to make loop(s). Correlation analysis of the two single-nucleosome movements [G (green) and R (red); their hypothetical movements are shown on the right] can be used to distinguish between these two possible models in living cells. ( C ) Scheme for Alexa488-dUTP (green) and Cy3-dCTP (red) incorporations during early DNA replication for dual-color labeling of nucleosomes. (D) Schematic for dual-color imaging with a beam splitter system (W-VIEW GEMINI, Hamamatsu Photonics). The images of two single-nucleosomes with different colors were acquired with a single sCMOS camera (left half, green color; right half, red color). ( E ) Representative images of single-nucleosomes labeled with Alexa488-dUTP (left) or Cy3-dCTP (right) in a living HeLa nucleus. The bar represents 1 μm. The box regions are enlarged to the right. The bar represents 500 nm.

Article Snippet: For each assay, 200 ng of an enhanced green fluorescent protein (EGFP) gene polymerase chain reaction (PCR) fragment amplified by the Cy3 PCR labeling kit (PP-301S-CY3; Jena Bioscience) and the linearized plasmid (pGEM-T Easy plasmid digested with EcoRI) were resuspended in PBS buffer containing 300 ng of anti-Cy3 antibody (sc-166894; Santa Cruz Biotechnology).

Techniques: Labeling, Imaging

Journal: Science Advances

Article Title: Condensed but liquid-like domain organization of active chromatin regions in living human cells

doi: 10.1126/sciadv.adf1488

Figure Lengend Snippet: (A) Four representative trajectory sets of two nucleosomes labeled with green (Alexa488-dUTP) and red (Cy3-dCTP). While the upper two sets show trajectories of two closely localized nucleosomes, the lower two represent distant nucleosomes. See also movies S5 to S8. (B) Histograms of congruence coefficient r c calculated between the two single-nucleosomes (green and red) whose averaged distances were in the ranges of 0 to 50 nm (median r c = 0.75), 50 to 100 nm (median r c = 0.48), 100 to 150 nm (median r c = 0.29), 150 to 200 nm (median r c = 0.08), and over 200 nm (median r c = 0.05) for 500 ms and the random control (median r c = 0.02). (C) Frequency plots of nucleosome pairs (green and red) with r c > 0.6 versus the pair distances. The dashed line shows the frequency of randomly collected nucleosome pairs with r c > 0.6. (D) Model of nucleosomes formed into a condensed domain with ~150 nm diameter.

Article Snippet: For each assay, 200 ng of an enhanced green fluorescent protein (EGFP) gene polymerase chain reaction (PCR) fragment amplified by the Cy3 PCR labeling kit (PP-301S-CY3; Jena Bioscience) and the linearized plasmid (pGEM-T Easy plasmid digested with EcoRI) were resuspended in PBS buffer containing 300 ng of anti-Cy3 antibody (sc-166894; Santa Cruz Biotechnology).

Techniques: Labeling, Control

Journal: Science Advances

Article Title: Condensed but liquid-like domain organization of active chromatin regions in living human cells

doi: 10.1126/sciadv.adf1488

Figure Lengend Snippet: ( A ) Schematic for two-point MSD. ( B ) Two-point MSD plots (±SD among ≥3 clusters) between Cy3- and Alexa488-nucleosomes whose averaged distances ( d ) were 0 to 50 nm in living siRAD21-treated HeLa cells (red, 54 pairs), untreated cells (black, 84 pairs), and TSA-treated cells (blue, 42 pairs). Ten or more pairs were grouped as a cluster. ( C ) Two-point MSD plots (±SD among ≥3 clusters; d , 50 to 150 nm) in living siRAD21-treated HeLa cells (red, 33 pairs), untreated cells (black, 95 pairs), and TSA-treated cells (blue, 35 pairs). ( D ) Schematic for replication foci labeled with Cy3-dCTP at high concentrations (left) and their representative images in a living HeLa nucleus (right). The square region is magnified at lower right. Note that each dot corresponds to one replication focus, consisting of one or a few contact domains. Also see movie S15. ( E ) Schematic for single-nucleosome imaging and their typical representation. Note that each dot corresponds to a single-nucleosome. ( F ) MSD plots (±SD among cells) of early replication foci (light blue, n = 12 cells) and single-nucleosomes at the early replicated regions (orange, n = 19 cells) in living HeLa cells. ** P < 0.001 by Kolmogorov-Smirnov test for foci versus single nucleosomes ( P = 4.8 × 10 −4 ). ( G ) Schematic for FRAP. ( H ) Representative images of Cy3-labeled early replication foci before, just after and 30 min after photobleaching. Photobleached region is shown by a yellow dotted line. XY -movement of the cell is corrected. ( I ) Mean normalized fluorescence intensity over time (fluorescence recovery curve) for Cy3-labeled early replication foci, n = 10 cells. ( J ) Mean normalized fluorescence intensity of photobleached region before, just after, and 30 min after photobleaching ( n = 11 cells). Also see movie S16.

Article Snippet: For each assay, 200 ng of an enhanced green fluorescent protein (EGFP) gene polymerase chain reaction (PCR) fragment amplified by the Cy3 PCR labeling kit (PP-301S-CY3; Jena Bioscience) and the linearized plasmid (pGEM-T Easy plasmid digested with EcoRI) were resuspended in PBS buffer containing 300 ng of anti-Cy3 antibody (sc-166894; Santa Cruz Biotechnology).

Techniques: Labeling, Imaging, Fluorescence

Journal: Zoological science

Article Title: Sponge cytogenetics - mitotic chromosomes of ten species of freshwater sponge.

doi: 10.2108/zsj.25.480

Figure Lengend Snippet: Fig. 4. FISH pattern of the ribosomal RNA genes and telomere (TTAGGG)n repeat sequences in E. fluviatilis (TON). (a) PI-stained metaphase spread hybridized with biotin-labeled 18S-28S ribosomal DNA as a probe. (b) DAPI-stained metaphase spread hybridized with the Cy3-labeled TTAGGG repeat sequence as a probe and (c) DAPI staining of the same metaphase. Arrowheads show the 18S-28S ribosomal DNA signals. Scale bar represents 5 μm.

Article Snippet: After hybridization, the slides were incubated with FITC-avidin (Roche Diagnostics) and mouse monoclonal antidigoxigenin clone DI-22 ascites fluid (Sigma-Aldrich) labeled with Cy3 by using a Cy3 Ab Labeling Kit (Amersham Biosciences) to stain the rDNA probe and the telomere repeat probe, respectively.

Techniques: Staining, Labeling, Sequencing

Journal: bioRxiv

Article Title: Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

doi: 10.1101/2020.04.03.003178

Figure Lengend Snippet: Genomic locations of the (a) Tbx3 and (b) Nanog genes annotated with the locations to which dCas9-GFP was targeted using either CARGO vectors or a single gRNA that targets nearby genomic repeats (red lines). Locations of bacterial artificial chromosome (BAC) DNA FISH probes for the Tbx3 and Nanog enhancers are also indicated as are the targeted enhancers themselves (green). The corresponding ChIP-seq profiles indicate the binding of the NuRD complex subunits CHD4 and MBD3 as well as the location of active enhancers (determined from the ChIP-seq profiles for H3K27ac, H3K4me1 and p300). (c) Representative confocal images showing co-localisation of dCas9-GFP (labelled using AF488-tagged anti-GFP nanobody) and AF647-BAC DNA FISH probes targeting the Nanog enhancer. (d) Representative confocal images of Cy3-labelled BAC DNA FISH probes targeting the Tbx3 promoter and AF647-labelled BAC DNA FISH probes targeting the Tbx3 enhancer. (e) Boxplots showing the enhancer-promoter distances in MBD3-inducible ESCs with and without tamoxifen: +MBD3 (blue) and -MBD3 ESCs (orange) respectively. There is a significant increase in enhancer-promoter distance in the presence of intact NuRD for Tbx3 (+MBD3, n = 70; -MBD3, n = 101), Bmp4 (+MBD3, n = 172; -MBD3, n = 71) and Sox2 (+MBD3, n = 42; -MBD3, n = 50) (**p<0.01, ***p < 10 −5 , Kolmogorov-Smirnov test). To estimate the precision limit of the experiment, control samples were generated in which the distance was measured for the Sox2 enhancer labelled with both Cy3 and AF647 (grey, n = 32).

Article Snippet: BAC DNA was labelled using Cy3 and Alexa Fluor 647 Nick Translation Labelling Kits (Jena Bioscience, PP-305S-CY3, PP-305S-AF647) and purified as previously described .

Techniques: ChIP-sequencing, Binding Assay, Generated

Journal: bioRxiv

Article Title: Restoring Shugoshin 1 reduces chromosome errors in human eggs

doi: 10.64898/2026.01.08.698387

Figure Lengend Snippet: (A) Representative immunofluorescence images showing the localization nascent transcripts (EU, green) inside the nucleus (blue) of early GV oocytes. Images show EU levels in negative control (no EU), control (with EU), upon treatment with α-amanitin, and triptolide. Scale bar: 10 µm. (B) Frequency of PSSC with α-amanitin measured by scoring the percentage of eggs with PSSC, similar to levels observed with triptolide . Plots show number of eggs analyzed on top. Statistical significance is measured by Fisher’s exact test, ns = not significant. (C) Representative time-lapse images showing the localization of MajSat RNA in GV and MI oocytes and MII eggs upon microinjection of GV oocytes with UTP-X-Cy3 labeled MajSat (top panel) and control (bottom panel) RNA. MajSat RNA localizes to the pericentromeres during metaphase I and metaphase II stages along with foci present in the cytoplasm. Cy3-labeled control RNA localizes only in the cytoplasm and not on chromosomes. MajSat and control RNA (green), chromosomes (H2B-SNAP, blue) are shown. Scale bars: 10 µm. (D) Co-localization of MajSat RNA and SGO1 observed on metaphase I chromosome spreads upon performing RNA FISH with immunofluorescence. Scale bar: 10 µm.

Article Snippet: The HighYield T7 Cy3 RNA Labelling Kit (Jena Bioscience, #RNT-101-CY3) was used to label amplified sequences with Cy3-labelled UTP (UTP-X-Cy3, 35%), followed by DNA removal using TurboTM DNAse (ThermoFisher).

Techniques: Immunofluorescence, Negative Control, Control, Microinjection, Labeling

Journal: Molecular Pharmaceutics

Article Title: Optimizing the Delivery of mRNA to Mesenchymal Stem Cells for Tissue Engineering Applications

doi: 10.1021/acs.molpharmaceut.3c00898

Figure Lengend Snippet: Distribution of mRNA nanoparticles in a collagen-nHA scaffold. (A) Representative confocal scanning micrograph of Cy3-labeled mRNA nanoparticles (jetPEI-modRNA 5 μg 2:1 v/w) in a cross section of a collagen-nHA scaffold. mRNA nanoparticles are fluorescently tagged (red), and the collagen-nHA scaffold structure autofluoresces (blue). mRNA nanoparticles remained close to both scaffold surfaces, where they had initially been soak-loaded. Scale bar = 500 μm. (B) Higher-magnification confocal scanning micrograph of (i) Cy3-labeled mRNA nanoparticles within the nanoparticle-rich area of the scaffold demonstrating even distribution of nanoparticles throughout the scaffold pores. (ii) A blank collagen-nHA scaffold was imaged as a control. Scale bar = 100 μm.

Article Snippet: To assess the distribution of mRNA nanoparticles within a collagen-nHA scaffold, modRNA was tagged with Cy3 using a Mirus Label IT Cy3 nucleic acid labeling kit (Medical Supply Company, Ireland) according to the manufacturer’s instructions.

Techniques: Labeling