snap cell tmr star  (New England Biolabs)


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    SNAP Cell TMR STAR
    Description:
    SNAP Cell TMR STAR 30 nmol
    Catalog Number:
    s9105s
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    344
    Size:
    30 nmol
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    Fluorochromes
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    New England Biolabs snap cell tmr star
    SNAP Cell TMR STAR
    SNAP Cell TMR STAR 30 nmol
    https://www.bioz.com/result/snap cell tmr star/product/New England Biolabs
    Average 99 stars, based on 75 article reviews
    Price from $9.99 to $1999.99
    snap cell tmr star - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin"

    Article Title: The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1219

    OTUD5 and UBR5 regulates the deposition of new Histone H2A at the damage sites. ( A ) Enlargement of SPT16 foci in UBR5 and OTUD5 knockdown cells. HeLa cells were irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters and fixed 1 hour after. The number of pixels in each γH2AX and SPT16 co-localized foci was measured using Image J, with each pixel represents an area of 0.2 μm 2 . The ratio of SPT16 to γH2AX was calculated by dividing the SPT16 area by the γH2AX area. The assay was performed in triplicates ( N = 35). Red bars indicate the median value for each set. ( B ) Ratio of SPT16 to Fok1 area in pTuner263 cells transfected with the siRNAs were quantified as in ( A ) The assay was performed in triplicates, N = 25. ( C ) Schematic for SNAP-H2A labeling and nucleosome integration. ( D ) U2OS cells stably expressing SNAP-H2A were first blocked with TMR Block and then irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters. Cells were labeled with TMR Star (red) and then fixed at indicated time points. Cells were counter-stained for γH2AX. The assay was performed in triplicates. Vector quantification of RFI of TMR signal (SNAP-H2A) ( N = 20); see ‘Materials and Methods’ section for details. Scale bars indicate 10 μm. (**** indicates P -value
    Figure Legend Snippet: OTUD5 and UBR5 regulates the deposition of new Histone H2A at the damage sites. ( A ) Enlargement of SPT16 foci in UBR5 and OTUD5 knockdown cells. HeLa cells were irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters and fixed 1 hour after. The number of pixels in each γH2AX and SPT16 co-localized foci was measured using Image J, with each pixel represents an area of 0.2 μm 2 . The ratio of SPT16 to γH2AX was calculated by dividing the SPT16 area by the γH2AX area. The assay was performed in triplicates ( N = 35). Red bars indicate the median value for each set. ( B ) Ratio of SPT16 to Fok1 area in pTuner263 cells transfected with the siRNAs were quantified as in ( A ) The assay was performed in triplicates, N = 25. ( C ) Schematic for SNAP-H2A labeling and nucleosome integration. ( D ) U2OS cells stably expressing SNAP-H2A were first blocked with TMR Block and then irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters. Cells were labeled with TMR Star (red) and then fixed at indicated time points. Cells were counter-stained for γH2AX. The assay was performed in triplicates. Vector quantification of RFI of TMR signal (SNAP-H2A) ( N = 20); see ‘Materials and Methods’ section for details. Scale bars indicate 10 μm. (**** indicates P -value

    Techniques Used: Irradiation, Transfection, Labeling, Stable Transfection, Expressing, Blocking Assay, Staining, Plasmid Preparation

    2) Product Images from "Constitutive centromere-associated network contacts confer differential stability on CENP-A nucleosomes in vitro and in the cell"

    Article Title: Constitutive centromere-associated network contacts confer differential stability on CENP-A nucleosomes in vitro and in the cell

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-10-0596

    CENP-C and CENP-N degradation has no significant effect on centromeric CENP-A maintenance. (A, B) Cell lines containing AID tagged CENP-N, CENP-C, or both were treated with IAA to degrade the indicated proteins. Blue bars represent cells not treated with IAA. Cells were maintained in IAA beginning just after mitosis (red bars) and harvested in an early S-phase thymidine (thym) arrest (A) or after mitosis (mustard bars) or in early S phase (green bars) and harvested in a G2-phase roscovitine (rosco) arrest (B). Centromeric CENP-A immunofluorescence signal was normalized to the no-IAA signal. (C) Degradation of CENP-N inhibits new CENP-A assembly but not preexisting CENP-A in chromatin. The CENP-N AID-sfGFP cell line containing a stably integrated SNAP-tagged CENP-A was either fluorescently labeled or quenched according to the schematic (left panel). Green bars represent the time of synthesis of the fluorescent population of SNAP-tagged CENP-A. IAA was added as in A. Centromeric TMR-Star intensity represents the fluorescent population of SNAP-tagged CENP-A. Data are presented as mean ± SEM for three independent replicates. * p
    Figure Legend Snippet: CENP-C and CENP-N degradation has no significant effect on centromeric CENP-A maintenance. (A, B) Cell lines containing AID tagged CENP-N, CENP-C, or both were treated with IAA to degrade the indicated proteins. Blue bars represent cells not treated with IAA. Cells were maintained in IAA beginning just after mitosis (red bars) and harvested in an early S-phase thymidine (thym) arrest (A) or after mitosis (mustard bars) or in early S phase (green bars) and harvested in a G2-phase roscovitine (rosco) arrest (B). Centromeric CENP-A immunofluorescence signal was normalized to the no-IAA signal. (C) Degradation of CENP-N inhibits new CENP-A assembly but not preexisting CENP-A in chromatin. The CENP-N AID-sfGFP cell line containing a stably integrated SNAP-tagged CENP-A was either fluorescently labeled or quenched according to the schematic (left panel). Green bars represent the time of synthesis of the fluorescent population of SNAP-tagged CENP-A. IAA was added as in A. Centromeric TMR-Star intensity represents the fluorescent population of SNAP-tagged CENP-A. Data are presented as mean ± SEM for three independent replicates. * p

    Techniques Used: Immunofluorescence, Stable Transfection, Labeling

    3) Product Images from "Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3"

    Article Title: Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3

    Journal: Journal of Virology

    doi: 10.1128/JVI.00477-18

    (A) Cell lysates from stable CHIKV cells. Live cells were stained with BG-TMR-Star and lysed with Glasgow lysis buffer. The lysate was then bound to plastic chamber slides overnight and imaged the following day. Images were acquired with an LSM880 microscope operated in Fast Airyscan mode. Cell lysates from uninfected, naive HuH-7 cells are shown as a control. (B) Zoomed-in views of the sample shown in panel A, providing higher-magnification views of nsP3-containing granules and corresponding bright-field images (transmission). (C) Cell lysates from HuH-7 cells infected with CHIKV ZsGreen-P3 . Samples were prepared as described for panel A and imaged in the green channel. (D) Schematic overview of different populations (P1 to P4) obtained during culture of stable CHIKV cells. (E) Wide-field microscopy of stable CHIKV cells passaged for 1 week in the absence of puromycin (P2). Naive HuH-7 cells and cells treated for 1 week with puromycin (P1) are shown as controls. Images were obtained with an IncuCyte Zoom live-cell imaging system. (F) Effect of puromycin treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. Confluence of the two populations was determined from images taken with an IncuCyte Zoom live-cell imaging system. (G) Effect of sodium arsenite treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. To induce cellular stress granules, sodium arsenite was added for at least 30 min. Cells were fixed and then stained for SNAP-nsP3 (cyan) and G3BP2 (magenta). Stained cells were imaged by Airyscan microscopy. FOVs 1 and 3 were centered on cells expressing ZsGreen, whereas FOVs 2 and 4 focused on cells that were ZsGreen negative.
    Figure Legend Snippet: (A) Cell lysates from stable CHIKV cells. Live cells were stained with BG-TMR-Star and lysed with Glasgow lysis buffer. The lysate was then bound to plastic chamber slides overnight and imaged the following day. Images were acquired with an LSM880 microscope operated in Fast Airyscan mode. Cell lysates from uninfected, naive HuH-7 cells are shown as a control. (B) Zoomed-in views of the sample shown in panel A, providing higher-magnification views of nsP3-containing granules and corresponding bright-field images (transmission). (C) Cell lysates from HuH-7 cells infected with CHIKV ZsGreen-P3 . Samples were prepared as described for panel A and imaged in the green channel. (D) Schematic overview of different populations (P1 to P4) obtained during culture of stable CHIKV cells. (E) Wide-field microscopy of stable CHIKV cells passaged for 1 week in the absence of puromycin (P2). Naive HuH-7 cells and cells treated for 1 week with puromycin (P1) are shown as controls. Images were obtained with an IncuCyte Zoom live-cell imaging system. (F) Effect of puromycin treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. Confluence of the two populations was determined from images taken with an IncuCyte Zoom live-cell imaging system. (G) Effect of sodium arsenite treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. To induce cellular stress granules, sodium arsenite was added for at least 30 min. Cells were fixed and then stained for SNAP-nsP3 (cyan) and G3BP2 (magenta). Stained cells were imaged by Airyscan microscopy. FOVs 1 and 3 were centered on cells expressing ZsGreen, whereas FOVs 2 and 4 focused on cells that were ZsGreen negative.

    Techniques Used: Staining, Lysis, Microscopy, Transmission Assay, Infection, Live Cell Imaging, Expressing

    4) Product Images from "A novel role for the histone acetyltransferase Hat1 in the CENP-A/CID assembly pathway in Drosophila melanogaster"

    Article Title: A novel role for the histone acetyltransferase Hat1 in the CENP-A/CID assembly pathway in Drosophila melanogaster

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1235

    Hat1 knock-down causes decreased CENP-A loading. ( A ) Experimental design of pulse labeling-mediated analysis of CENP-A centromeric loading. ( B ) RNAi efficiency was determined by RT-qPCR for Hat1. Transcript levels for Hat1, CENP-A and histone H4 in Hat1 RNAi cells were normalized against GAPDH and are expressed relative to those of the respective genes in TetR-treated S2 cells (control). ( C ) Example image of reduced CENP-A intensities in Hat1 RNAi cells. Cells were processed according to the scheme in (A) and newly loaded SNAP-CENP-A was visualized by staining with TMR-Star. Images were acquired and processed with identical settings. ( D ) Quantification of SNAP-CENP-A intensities in Hat1 knock-down and TetR RNAi control cells using Imaris v5.1 software. Statistical significance was determined by unpaired t -test and Mann–Whitney test (*** P
    Figure Legend Snippet: Hat1 knock-down causes decreased CENP-A loading. ( A ) Experimental design of pulse labeling-mediated analysis of CENP-A centromeric loading. ( B ) RNAi efficiency was determined by RT-qPCR for Hat1. Transcript levels for Hat1, CENP-A and histone H4 in Hat1 RNAi cells were normalized against GAPDH and are expressed relative to those of the respective genes in TetR-treated S2 cells (control). ( C ) Example image of reduced CENP-A intensities in Hat1 RNAi cells. Cells were processed according to the scheme in (A) and newly loaded SNAP-CENP-A was visualized by staining with TMR-Star. Images were acquired and processed with identical settings. ( D ) Quantification of SNAP-CENP-A intensities in Hat1 knock-down and TetR RNAi control cells using Imaris v5.1 software. Statistical significance was determined by unpaired t -test and Mann–Whitney test (*** P

    Techniques Used: Labeling, Quantitative RT-PCR, Staining, Software, MANN-WHITNEY

    5) Product Images from "Visualizing the dynamic coupling of claudin strands to the actin cytoskeleton through ZO-1"

    Article Title: Visualizing the dynamic coupling of claudin strands to the actin cytoskeleton through ZO-1

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E16-10-0698

    Pulse-chase-pulse experiments indicate new cldn addition to strands is focal and concentrates at sites of strand breaks. (A) Diagram of pulse-chase-pulse method. (B) Immunofluorescence images of SNAP-tagged cldn labeled with TMR only (left), TMR, block, and after 3, 5, and 8 h with SNAP- Cell 647 (middle three consecutive panels), and with SNAP-Cell 647 only (right; bar, 5 μm). (C) Higher-magnification images from 3-h pulse-chase-pulse experiment; arrows point to sites of addition of newly synthesized cldns. (D) Models for possible mechanisms of cldn addition to new strands.
    Figure Legend Snippet: Pulse-chase-pulse experiments indicate new cldn addition to strands is focal and concentrates at sites of strand breaks. (A) Diagram of pulse-chase-pulse method. (B) Immunofluorescence images of SNAP-tagged cldn labeled with TMR only (left), TMR, block, and after 3, 5, and 8 h with SNAP- Cell 647 (middle three consecutive panels), and with SNAP-Cell 647 only (right; bar, 5 μm). (C) Higher-magnification images from 3-h pulse-chase-pulse experiment; arrows point to sites of addition of newly synthesized cldns. (D) Models for possible mechanisms of cldn addition to new strands.

    Techniques Used: Pulse Chase, Immunofluorescence, Labeling, Blocking Assay, Synthesized

    6) Product Images from "CENP-A modifications on Ser68 and Lys124 are dispensable for establishment, maintenance, and long-term function of human centromeres"

    Article Title: CENP-A modifications on Ser68 and Lys124 are dispensable for establishment, maintenance, and long-term function of human centromeres

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2016.12.014

    Expression of CENP-A S68Q or CENP-A K124R (A) Schematic of the biallelic gene replacement approach used to replace the endogenous CENP-A gene with EGFP-AID-tagged CENP-A on one allele and CENP-A (wild type or mutant) tagged with SNAP-3xHA-P2A-NeoR on the other allele in DLD-1 TIR-1 cells via CRISPR/Cas9-mediated gene editing. (B) Schematic showing the indicated mutants of CENP-A tagged with SNAP-3xHA-P2A-NeoR, which replace the endogenous CENP-A gene on one allele, as indicated in Panel A. (C) Immunoblot of whole cell lysates from each of the indicated cell lines. Relevant cell lines were treated with 500 μM IAA for 24 hr to degrade EGFP-AID-tagged CENP-A. The blot was probed with anti-CENP-A and anti-tubulin antibodies. (D) Representative images showing localization of EGFP-AID-tagged CENP-A and CENP-A(wild type or mutant)-SNAP-3xHA at centromeres. Upon treatment with 500 μM IAA for 24 hr, the EGFP-AID-tagged CENP-A is no longer detected. (E) Quantification of the percentage of viable cells in the indicated cell lines upon treatment with 500 μM IAA for 8 d. Every 2 d, cells were collected and stained with Trypan Blue and counted on a hemocytometer to calculate the percentage of viable cells based on Trypan Blue uptake. Mean +/− SEM is shown for each time point. (F) Representative images of the indicated cell lines after 8 d of treatment with 500 μM IAA. The SNAP-3xHA-tagged CENP-A mutants are still present at endogenous centromeres. (G) Schematic for the quench-chase-pulse experiment in which the existing pool of CENP-A is quenched with SNAP-Cell Block, new CENP-A is synthesized, and newly loaded CENP-A is labeled with TMR- Star 24 hr later. (H) Quantification of the quench-chase-pulse experiment in which TMR- Star and total CENP-A signals are measured at centromeres in G1 cells (marked by a tubulin midbody remnant). Mean +/− SEM is shown. (I) Representative images showing that TMR- Star -labeled CENP-A is loaded at centromeres for each of the cell lines. The tubulin midbody remnant is shown between daughter G1 cells. Cells in which TMR- Star -labeled CENP-A is not detected at centromeres are shown in each representative image. Scale bar: 5 μm. Insets show magnification of the boxed region.
    Figure Legend Snippet: Expression of CENP-A S68Q or CENP-A K124R (A) Schematic of the biallelic gene replacement approach used to replace the endogenous CENP-A gene with EGFP-AID-tagged CENP-A on one allele and CENP-A (wild type or mutant) tagged with SNAP-3xHA-P2A-NeoR on the other allele in DLD-1 TIR-1 cells via CRISPR/Cas9-mediated gene editing. (B) Schematic showing the indicated mutants of CENP-A tagged with SNAP-3xHA-P2A-NeoR, which replace the endogenous CENP-A gene on one allele, as indicated in Panel A. (C) Immunoblot of whole cell lysates from each of the indicated cell lines. Relevant cell lines were treated with 500 μM IAA for 24 hr to degrade EGFP-AID-tagged CENP-A. The blot was probed with anti-CENP-A and anti-tubulin antibodies. (D) Representative images showing localization of EGFP-AID-tagged CENP-A and CENP-A(wild type or mutant)-SNAP-3xHA at centromeres. Upon treatment with 500 μM IAA for 24 hr, the EGFP-AID-tagged CENP-A is no longer detected. (E) Quantification of the percentage of viable cells in the indicated cell lines upon treatment with 500 μM IAA for 8 d. Every 2 d, cells were collected and stained with Trypan Blue and counted on a hemocytometer to calculate the percentage of viable cells based on Trypan Blue uptake. Mean +/− SEM is shown for each time point. (F) Representative images of the indicated cell lines after 8 d of treatment with 500 μM IAA. The SNAP-3xHA-tagged CENP-A mutants are still present at endogenous centromeres. (G) Schematic for the quench-chase-pulse experiment in which the existing pool of CENP-A is quenched with SNAP-Cell Block, new CENP-A is synthesized, and newly loaded CENP-A is labeled with TMR- Star 24 hr later. (H) Quantification of the quench-chase-pulse experiment in which TMR- Star and total CENP-A signals are measured at centromeres in G1 cells (marked by a tubulin midbody remnant). Mean +/− SEM is shown. (I) Representative images showing that TMR- Star -labeled CENP-A is loaded at centromeres for each of the cell lines. The tubulin midbody remnant is shown between daughter G1 cells. Cells in which TMR- Star -labeled CENP-A is not detected at centromeres are shown in each representative image. Scale bar: 5 μm. Insets show magnification of the boxed region.

    Techniques Used: Expressing, Mutagenesis, CRISPR, Staining, Blocking Assay, Synthesized, Labeling

    7) Product Images from "High-resolution visualization of H3 variants during replication reveals their controlled recycling"

    Article Title: High-resolution visualization of H3 variants during replication reveals their controlled recycling

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05697-1

    Tracking histone H3 variants with STORM microscopy. a Labeling scheme using H3.3- or H3.1-SNAP to follow global (top) or parental (bottom) histones. A pulse using the fluorophore TMR (orange) labels SNAP-tagged H3.3 or H3.1. EdU incorporation at the end of the assay allows the detection of replicated DNA (green). This EdU labeling is carried out either during the TMR pulse to compare global H3 distribution with patches of DNA synthesis or after a chase period that allows synthesis and deposition of new unlabeled H3.3- or H3.1-SNAP. The latter enables the localization of 48h-old parental histones with new patches of DNA synthesis. In all cases, we eliminate soluble histones by Triton extraction prior to fixation in order to analyze chromatin-bound H3.3 or H3.1 fractions. b Left panels: confocal images of global H3.1 (TMR, red) and replicated DNA (EdU, green) at different S-phase stages and outside S phase. Cells outside S phase are EdU negative, early S phase shows patterns broadly labeling the nucleus with the exception of the nucleoli and mid/late S phase shows patterns with clear enrichment at the nuclear periphery and around nucleoli. Right panels: STORM images of global H3.1 (TMR, orange) and replicated DNA (EdU, green) in cells outside S phase, early S phase and mid/late S phase. We used the ViSP software to render STORM images. Scale bars represent 10 μm
    Figure Legend Snippet: Tracking histone H3 variants with STORM microscopy. a Labeling scheme using H3.3- or H3.1-SNAP to follow global (top) or parental (bottom) histones. A pulse using the fluorophore TMR (orange) labels SNAP-tagged H3.3 or H3.1. EdU incorporation at the end of the assay allows the detection of replicated DNA (green). This EdU labeling is carried out either during the TMR pulse to compare global H3 distribution with patches of DNA synthesis or after a chase period that allows synthesis and deposition of new unlabeled H3.3- or H3.1-SNAP. The latter enables the localization of 48h-old parental histones with new patches of DNA synthesis. In all cases, we eliminate soluble histones by Triton extraction prior to fixation in order to analyze chromatin-bound H3.3 or H3.1 fractions. b Left panels: confocal images of global H3.1 (TMR, red) and replicated DNA (EdU, green) at different S-phase stages and outside S phase. Cells outside S phase are EdU negative, early S phase shows patterns broadly labeling the nucleus with the exception of the nucleoli and mid/late S phase shows patterns with clear enrichment at the nuclear periphery and around nucleoli. Right panels: STORM images of global H3.1 (TMR, orange) and replicated DNA (EdU, green) in cells outside S phase, early S phase and mid/late S phase. We used the ViSP software to render STORM images. Scale bars represent 10 μm

    Techniques Used: Microscopy, Labeling, DNA Synthesis, Software

    8) Product Images from "POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication"

    Article Title: POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2018.08.043

    Transient Depletion of the POLE3-POLE4 Complex Affects Histone Deposition in SNAP Tag H3.1-Expressing Cells (A) In HeLa H3.1-SNAP: at left, quench-chase-pulse experiment to follow new H3.1; at right, pulse-chase experiment to follow parental H3.1. A quenching step labels all pre-existing histones with a non-fluorescent dye. A chase step allows synthesis and deposition of new unlabeled H3.1-SNAP. A pulse using the fluorophore TMR (red) labels available H3.1-SNAP. EdU incorporation at the end of the assay allows the detection of replicated DNA (green). A Triton extraction step is performed prior to fixation to eliminate soluble histones and analyze chromatin-bound H3.1. (B) Representative images of new (left) or parental (right) H3.1 (TMR, red) and replication sites (EdU, green) in control, POLE1-depleted, POLE3-depleted, or POLE4-depleted conditions. Scale bars, 10 μm. (C) Quantification of TMR fluorescence signal per nucleus normalized to control mean for new (left) or parental (right) H3.1 in control, POLE1-depleted, POLE3-depleted, or POLE4-depleted conditions (n = 3). For new H3.1 (left), only cells in S phase were quantified.
    Figure Legend Snippet: Transient Depletion of the POLE3-POLE4 Complex Affects Histone Deposition in SNAP Tag H3.1-Expressing Cells (A) In HeLa H3.1-SNAP: at left, quench-chase-pulse experiment to follow new H3.1; at right, pulse-chase experiment to follow parental H3.1. A quenching step labels all pre-existing histones with a non-fluorescent dye. A chase step allows synthesis and deposition of new unlabeled H3.1-SNAP. A pulse using the fluorophore TMR (red) labels available H3.1-SNAP. EdU incorporation at the end of the assay allows the detection of replicated DNA (green). A Triton extraction step is performed prior to fixation to eliminate soluble histones and analyze chromatin-bound H3.1. (B) Representative images of new (left) or parental (right) H3.1 (TMR, red) and replication sites (EdU, green) in control, POLE1-depleted, POLE3-depleted, or POLE4-depleted conditions. Scale bars, 10 μm. (C) Quantification of TMR fluorescence signal per nucleus normalized to control mean for new (left) or parental (right) H3.1 in control, POLE1-depleted, POLE3-depleted, or POLE4-depleted conditions (n = 3). For new H3.1 (left), only cells in S phase were quantified.

    Techniques Used: Expressing, Pulse Chase, Fluorescence

    9) Product Images from "Dual Bioorthogonal Labeling of the Amyloid-β Protein Precursor Facilitates Simultaneous Visualization of the Protein and Its Cleavage Products"

    Article Title: Dual Bioorthogonal Labeling of the Amyloid-β Protein Precursor Facilitates Simultaneous Visualization of the Protein and Its Cleavage Products

    Journal: Journal of Alzheimer's Disease

    doi: 10.3233/JAD-190898

    A) Confocal microscopy of HEK293T cells verifies dual labeling of the expressed AβPP-SNAP protein. HEK293T cells were transfected with the amber codon machinery and AβPP H13 > amber or wt. Live cells were labeled with TMR-Star (red) and mT-BDP-FL, which reacts with the ncAA (green). Afterwards, the nuclei were labeled with SiR-HOECHST. Labeled cells were imaged sequentially by confocal microscopy (40× magnification). B, C) Mean intensity of mT-BDP-FL and TMR per cell in each image. Number of experiments = 3. Five pictures were taken in each well.
    Figure Legend Snippet: A) Confocal microscopy of HEK293T cells verifies dual labeling of the expressed AβPP-SNAP protein. HEK293T cells were transfected with the amber codon machinery and AβPP H13 > amber or wt. Live cells were labeled with TMR-Star (red) and mT-BDP-FL, which reacts with the ncAA (green). Afterwards, the nuclei were labeled with SiR-HOECHST. Labeled cells were imaged sequentially by confocal microscopy (40× magnification). B, C) Mean intensity of mT-BDP-FL and TMR per cell in each image. Number of experiments = 3. Five pictures were taken in each well.

    Techniques Used: Confocal Microscopy, Labeling, Transfection

    10) Product Images from "Reconstituting Drosophila Centromere Identity in Human Cells"

    Article Title: Reconstituting Drosophila Centromere Identity in Human Cells

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2019.08.067

    Drosophila dCENP-A Can Be Propagated at Ectopic Sites on Human Chromosomes (A) Experimental procedure to test epigenetic inheritance of dCENP-A in human cells. 48 h (day 2) after transfection of Cal1-GFP-LacI and dCENP-A-SNAP, cells were either stained with TMR ∗ and subjected to IF or incubated with IPTG to remove CAL1-GFP-LacI binding and SNAP-Cell Block to quench existing SNAP-dCENP-A molecules. After further culturing for 24 h, cells were incubated with TMR-Star to stain newly synthesized SNAP-dCENP-A (day 3). (B and C) Representative IF images of the recruitment of dCENP-A to the LacO arrays 2 days (B) or 3 days (C) after transfection of CAL1-GFP-LacIw and dCENP-A-SNAP in U2OS-LacO stable cell line expressing all three Drosophila centromeric proteins or only dCENP-A. Insets show magnification of the boxed regions. Scale bar, 5 μm. (D) Quantification of the percentage of cells with TMR ∗ signal at the LacO using imaging data of (B) and (C). Two experiments (dark circles) and their average (column) are shown. ∗ p
    Figure Legend Snippet: Drosophila dCENP-A Can Be Propagated at Ectopic Sites on Human Chromosomes (A) Experimental procedure to test epigenetic inheritance of dCENP-A in human cells. 48 h (day 2) after transfection of Cal1-GFP-LacI and dCENP-A-SNAP, cells were either stained with TMR ∗ and subjected to IF or incubated with IPTG to remove CAL1-GFP-LacI binding and SNAP-Cell Block to quench existing SNAP-dCENP-A molecules. After further culturing for 24 h, cells were incubated with TMR-Star to stain newly synthesized SNAP-dCENP-A (day 3). (B and C) Representative IF images of the recruitment of dCENP-A to the LacO arrays 2 days (B) or 3 days (C) after transfection of CAL1-GFP-LacIw and dCENP-A-SNAP in U2OS-LacO stable cell line expressing all three Drosophila centromeric proteins or only dCENP-A. Insets show magnification of the boxed regions. Scale bar, 5 μm. (D) Quantification of the percentage of cells with TMR ∗ signal at the LacO using imaging data of (B) and (C). Two experiments (dark circles) and their average (column) are shown. ∗ p

    Techniques Used: Transfection, Staining, Incubation, Binding Assay, Blocking Assay, Synthesized, Stable Transfection, Expressing, Imaging

    11) Product Images from "PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways"

    Article Title: PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

    Journal: Immunity

    doi: 10.1016/j.immuni.2019.11.003

    PD-L1 Binds CD80 in Cis , and Atezolizumab Disrupts this Interaction (A) Representative TIRF images of PD-L1 LUVs captured by PD-1 SLB, CD80 SLB, or CD86 SLB; each LUV is registered as a green spot. Bar graph summarizes the fluorescence intensity (FI) of the LUV channel under indicated conditions, normalized to the intensity of the condition with PD-1 SLBs. Data are means ± SEM, n = 3. Scale bars, 5 μm. (B) A FRET assay showing PD-L1:CD80 cis -interaction on cell membranes. Cartoons on the left depict a HEK293T cell co-expressing PD-L1 (labeled with CS547, donor) and either CD80 or CD86 (labeled with SSAF647, acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell at the indicated channels. Further right are calculated FRET efficiency images (pseudo-color; the yellow to purple spectrum denotes strong to weak FRET) and the differential interference contrast (DIC) images. Rightmost are bar graphs summarizing the FRET efficiencies as mean ± SEM, n > 25 cells from 3 independent experiments. Scale bars, 10 μm. (C) Same as (B) except replacing PD-L1 with PD-L2. (D) On the left is a cartoon depicting an LUV FRET assay for probing PD-L1:CD80 cis -interaction and atezolizumab (Atezo) effects. SC505 (donor) labeled SNAP-PD-L1-His was pre-bound to LUVs via DGS-NTA-Ni. Subsequently added TMR (acceptor) labeled SNAP-CD80-His bound to the LUVs and interacted with PD-L1 in cis , causing FRET and SC505 quenching (black trace). On the right are time courses of normalized SC505 fluorescence under the indicated conditions. Color coding is as follows: blue, same as black except plus atezolizumab; magenta, same as black except using TMR*CD80 lacking a His tag; orange, same as black except replacing PD-L1 with PD-L2; gray, same as black except presenting TMR*CD80 in trans . Data are representative of 3 independent replicates. Unpaired two-tailed Student’s t test: *p
    Figure Legend Snippet: PD-L1 Binds CD80 in Cis , and Atezolizumab Disrupts this Interaction (A) Representative TIRF images of PD-L1 LUVs captured by PD-1 SLB, CD80 SLB, or CD86 SLB; each LUV is registered as a green spot. Bar graph summarizes the fluorescence intensity (FI) of the LUV channel under indicated conditions, normalized to the intensity of the condition with PD-1 SLBs. Data are means ± SEM, n = 3. Scale bars, 5 μm. (B) A FRET assay showing PD-L1:CD80 cis -interaction on cell membranes. Cartoons on the left depict a HEK293T cell co-expressing PD-L1 (labeled with CS547, donor) and either CD80 or CD86 (labeled with SSAF647, acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell at the indicated channels. Further right are calculated FRET efficiency images (pseudo-color; the yellow to purple spectrum denotes strong to weak FRET) and the differential interference contrast (DIC) images. Rightmost are bar graphs summarizing the FRET efficiencies as mean ± SEM, n > 25 cells from 3 independent experiments. Scale bars, 10 μm. (C) Same as (B) except replacing PD-L1 with PD-L2. (D) On the left is a cartoon depicting an LUV FRET assay for probing PD-L1:CD80 cis -interaction and atezolizumab (Atezo) effects. SC505 (donor) labeled SNAP-PD-L1-His was pre-bound to LUVs via DGS-NTA-Ni. Subsequently added TMR (acceptor) labeled SNAP-CD80-His bound to the LUVs and interacted with PD-L1 in cis , causing FRET and SC505 quenching (black trace). On the right are time courses of normalized SC505 fluorescence under the indicated conditions. Color coding is as follows: blue, same as black except plus atezolizumab; magenta, same as black except using TMR*CD80 lacking a His tag; orange, same as black except replacing PD-L1 with PD-L2; gray, same as black except presenting TMR*CD80 in trans . Data are representative of 3 independent replicates. Unpaired two-tailed Student’s t test: *p

    Techniques Used: Fluorescence, Expressing, Labeling, Two Tailed Test

    Cis -PD-L1 Inhibits CD80:CTLA-4 Interaction through Disrupting CD80 Homodimers (A) Representative flow-cytometry histograms of CTLA-4-huFc staining of the indicated types of Raji cells. Bound CTLA-4-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CTLA-4-huFc). Shown in gray are Raji (CD80 + CD86 − ) cells stained by isolated huFc domain. Means ± SEM, n ≥ 3. (B) Representative flow-cytometry histograms of CTLA-4-moFc staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab (Atezo) (20 μg/mL). Bound moFc was labeled by AF647 anti-mouse IgG Fc, the MFI of which was plotted against (CTLA-4-moFc). Shown in gray are atezolizumab-treated Raji (CD80 + CD86 − PD-L1-mCherry + ) cells stained by isolated moFc domain. Means ± SEM, n ≥ 3. (C) Representative flow-cytometry histograms of CTLA-4-GCN4*SC647 staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab and of Raji (CD80 − CD86 − ) cells with atezolizumab. MFI of SC647 was plotted against the input concentration (means ± SEM, n ≥ 3). (D) At the top are flow-cytometry histograms showing both PD-L1 and CD80 amounts on a population of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with tight PD-L1 expression and a wide range of CD80 expression. The cells were stained with either phycoerythrin (PE) anti-CD80, PE anti-PD-L1, or PE isotype, and the 3 histograms overlaid. On the bottom is a flow-cytometry dot plot showing CTLA-4-GCN4*SC647 staining of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with or without atezolizumab. Gray dots correspond to control signals of unstained cells. CD80 + cells were gated by the vertical dash line, determined by the mGFP signal of parental Raji (CD80 − CD86 − ) cells. (E) A FRET assay probing CD80:CD80 homodimerization on cell membranes. In the first row, the leftmost cartoon depicts a HEK293T cell expressing SNAP-CD80, with a subpopulation labeled with SS549 (donor) and the rest labeled with SSAF647 (acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell. Further on the right is the calculated pseudo-color FRET efficiency image (yellow to purple spectrum denotes strong to weak FRET) and the DIC image. The second and third rows are the same as the first row except replacing SNAP-CD80 with SNAP-CD80 (I92R) or with SNAP-CD86. The fourth row is the same as the first row except with co-expressed unlabeled PD-L1. The fifth row is the same as fourth row except in the presence of atezolizumab. The bar graph summarizes the FRET efficiencies as mean ± SEM, n > 22 cells from 3 independent experiments. Scale bars, 10 μm. (F) An LUV FRET assay for probing CD80:CD80 homodimerization and PD-L1 effects. Shown is a representative time course of normalized FI of LUV-bound SC505*CD80-His, challenged by TMR*CD80-His and then by indicated concentrations of unlabeled PD-L1-His, with or without atezolizumab (Atezo) (20 μg/mL). (G) An LUV FRET assay showing that a single point mutation in CD80 disrupts both CD80:CD80 homodimerization and PD-L1:CD80 heterodimerization. Each indicated SC505 (energy donor)-labeled protein was pre-coupled to DGS-NTA-Ni containing LUVs through its His-tag, and challenged with TMR (energy acceptor)-labeled proteins as indicated. Shown are representative time courses of 3 independent replicates. (H) Representative TIRF images of Raji (CD80-mGFP + CD86 − PD-L1-SNAP + . Unpaired two-tailed Student’s for genotypes of cells related to this figure.
    Figure Legend Snippet: Cis -PD-L1 Inhibits CD80:CTLA-4 Interaction through Disrupting CD80 Homodimers (A) Representative flow-cytometry histograms of CTLA-4-huFc staining of the indicated types of Raji cells. Bound CTLA-4-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CTLA-4-huFc). Shown in gray are Raji (CD80 + CD86 − ) cells stained by isolated huFc domain. Means ± SEM, n ≥ 3. (B) Representative flow-cytometry histograms of CTLA-4-moFc staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab (Atezo) (20 μg/mL). Bound moFc was labeled by AF647 anti-mouse IgG Fc, the MFI of which was plotted against (CTLA-4-moFc). Shown in gray are atezolizumab-treated Raji (CD80 + CD86 − PD-L1-mCherry + ) cells stained by isolated moFc domain. Means ± SEM, n ≥ 3. (C) Representative flow-cytometry histograms of CTLA-4-GCN4*SC647 staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab and of Raji (CD80 − CD86 − ) cells with atezolizumab. MFI of SC647 was plotted against the input concentration (means ± SEM, n ≥ 3). (D) At the top are flow-cytometry histograms showing both PD-L1 and CD80 amounts on a population of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with tight PD-L1 expression and a wide range of CD80 expression. The cells were stained with either phycoerythrin (PE) anti-CD80, PE anti-PD-L1, or PE isotype, and the 3 histograms overlaid. On the bottom is a flow-cytometry dot plot showing CTLA-4-GCN4*SC647 staining of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with or without atezolizumab. Gray dots correspond to control signals of unstained cells. CD80 + cells were gated by the vertical dash line, determined by the mGFP signal of parental Raji (CD80 − CD86 − ) cells. (E) A FRET assay probing CD80:CD80 homodimerization on cell membranes. In the first row, the leftmost cartoon depicts a HEK293T cell expressing SNAP-CD80, with a subpopulation labeled with SS549 (donor) and the rest labeled with SSAF647 (acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell. Further on the right is the calculated pseudo-color FRET efficiency image (yellow to purple spectrum denotes strong to weak FRET) and the DIC image. The second and third rows are the same as the first row except replacing SNAP-CD80 with SNAP-CD80 (I92R) or with SNAP-CD86. The fourth row is the same as the first row except with co-expressed unlabeled PD-L1. The fifth row is the same as fourth row except in the presence of atezolizumab. The bar graph summarizes the FRET efficiencies as mean ± SEM, n > 22 cells from 3 independent experiments. Scale bars, 10 μm. (F) An LUV FRET assay for probing CD80:CD80 homodimerization and PD-L1 effects. Shown is a representative time course of normalized FI of LUV-bound SC505*CD80-His, challenged by TMR*CD80-His and then by indicated concentrations of unlabeled PD-L1-His, with or without atezolizumab (Atezo) (20 μg/mL). (G) An LUV FRET assay showing that a single point mutation in CD80 disrupts both CD80:CD80 homodimerization and PD-L1:CD80 heterodimerization. Each indicated SC505 (energy donor)-labeled protein was pre-coupled to DGS-NTA-Ni containing LUVs through its His-tag, and challenged with TMR (energy acceptor)-labeled proteins as indicated. Shown are representative time courses of 3 independent replicates. (H) Representative TIRF images of Raji (CD80-mGFP + CD86 − PD-L1-SNAP + . Unpaired two-tailed Student’s for genotypes of cells related to this figure.

    Techniques Used: Flow Cytometry, Cytometry, Staining, Labeling, Isolation, Concentration Assay, Expressing, Mutagenesis, Two Tailed Test

    12) Product Images from "The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance in Clostridium difficile"

    Article Title: The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance in Clostridium difficile

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00005-19

    Organization, localization, and kinase activity of the PrkC protein. (A) Organization of the domains of C. difficile CD2578-PrkC. PrkC contains a cytoplasmic kinase domain in the N-terminal part (brown), a transmembrane (TM) segment, two PASTA domains (green), and an atypical SGN (Ser, Gly, Asn)-rich domain in the C-terminal part (pink). The conserved lysine residue (K39) within the ATP-binding P loop of the kinase that is required for phosphotransfer is indicated. (B) Localization of the PrkC-HA-tagged protein. Cells expressing prkC fused to HA were grown in the presence of 15 ng/ml of ATc and harvested during exponential growth. Samples were fractionated into membrane (Mb) and cytoplasmic (Cy) fractions. Protein fractions were analyzed by Western blotting using an antibody raised against HA. (C) Localization of the SNAP-PrkC fusion during growth. The SNAP-PrkC protein was produced during the exponential growth phase in the presence of 50 ng/ml ATc. After labeling with the TMR-Star substrate, PrkC-SNAP localization was analyzed by fluorescence microscopy. AF, autofluorescence. The scale bar represents 5 μm. (D) Western blot performed after fractionation in a soluble fraction (lanes 1) and an insoluble fraction (lanes 2) obtained from exponential-phase cultures of the WT, the Δ prkC mutant, the complemented strain, and the Δ prkC mutant carrying pDIA6103- prkC -K39→A (K→A). An anti-P-Thr antibody was used to detect phosphorylated threonine. Red arrows indicate the bands detected in the WT strain that disappeared from the Δ prkC mutant.
    Figure Legend Snippet: Organization, localization, and kinase activity of the PrkC protein. (A) Organization of the domains of C. difficile CD2578-PrkC. PrkC contains a cytoplasmic kinase domain in the N-terminal part (brown), a transmembrane (TM) segment, two PASTA domains (green), and an atypical SGN (Ser, Gly, Asn)-rich domain in the C-terminal part (pink). The conserved lysine residue (K39) within the ATP-binding P loop of the kinase that is required for phosphotransfer is indicated. (B) Localization of the PrkC-HA-tagged protein. Cells expressing prkC fused to HA were grown in the presence of 15 ng/ml of ATc and harvested during exponential growth. Samples were fractionated into membrane (Mb) and cytoplasmic (Cy) fractions. Protein fractions were analyzed by Western blotting using an antibody raised against HA. (C) Localization of the SNAP-PrkC fusion during growth. The SNAP-PrkC protein was produced during the exponential growth phase in the presence of 50 ng/ml ATc. After labeling with the TMR-Star substrate, PrkC-SNAP localization was analyzed by fluorescence microscopy. AF, autofluorescence. The scale bar represents 5 μm. (D) Western blot performed after fractionation in a soluble fraction (lanes 1) and an insoluble fraction (lanes 2) obtained from exponential-phase cultures of the WT, the Δ prkC mutant, the complemented strain, and the Δ prkC mutant carrying pDIA6103- prkC -K39→A (K→A). An anti-P-Thr antibody was used to detect phosphorylated threonine. Red arrows indicate the bands detected in the WT strain that disappeared from the Δ prkC mutant.

    Techniques Used: Activity Assay, Binding Assay, Expressing, Western Blot, Produced, Labeling, Fluorescence, Microscopy, Fractionation, Mutagenesis

    13) Product Images from "Heat shock increases conjugation efficiency in Clostridium difficile"

    Article Title: Heat shock increases conjugation efficiency in Clostridium difficile

    Journal: Anaerobe

    doi: 10.1016/j.anaerobe.2016.06.009

    Precise manipulation of the R20291 genome accelerated by the use of optimised conjugation. (A) 0.8% Agarose gel showing PCR fragments amplified using primers flanking the SNAP-tag coding sequence insertion site in the chromosomal divIVA gene. The increase of approximately 550bp from 885 bp (R20291) to 1443 bp (R20291 divIVA-SNAP) suggests the correct insertion of the SNAP-tag coding sequence. (B) 12% SDS-PAGE gel imaged using a fluorescence imager, showing resolved lysates from SNAP TMR-star treated R20291 and R20291 divIVA-SNAP . A band at approximately 40 kDa in the mutant corresponds to the addition of a SNAP-tag on DivIVA. (C) Brightfield and (D) fluorescence microscopy of exponentially growing R20291 divIVA-SNAP stained with 250 nM SNAP TMR-star for 30 min, showing mostly septal and some polar localisation of fluorescence. This demonstrates successful modification of the R20291 genome. Scale bar represents 5 μm.
    Figure Legend Snippet: Precise manipulation of the R20291 genome accelerated by the use of optimised conjugation. (A) 0.8% Agarose gel showing PCR fragments amplified using primers flanking the SNAP-tag coding sequence insertion site in the chromosomal divIVA gene. The increase of approximately 550bp from 885 bp (R20291) to 1443 bp (R20291 divIVA-SNAP) suggests the correct insertion of the SNAP-tag coding sequence. (B) 12% SDS-PAGE gel imaged using a fluorescence imager, showing resolved lysates from SNAP TMR-star treated R20291 and R20291 divIVA-SNAP . A band at approximately 40 kDa in the mutant corresponds to the addition of a SNAP-tag on DivIVA. (C) Brightfield and (D) fluorescence microscopy of exponentially growing R20291 divIVA-SNAP stained with 250 nM SNAP TMR-star for 30 min, showing mostly septal and some polar localisation of fluorescence. This demonstrates successful modification of the R20291 genome. Scale bar represents 5 μm.

    Techniques Used: Conjugation Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Sequencing, SDS Page, Fluorescence, Mutagenesis, Microscopy, Staining, Modification

    14) Product Images from "Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings"

    Article Title: Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings

    Journal: bioRxiv

    doi: 10.1101/2020.03.09.983924

    SNAP-tag enabled in vivo imaging of tubulin in BY-2 cells and Arabidopsis a , Labeling mechanism of SNAP-tag. b , Cortical microtubules in SNAP-TUA5 expressing BY-2 cells with SNAP dyes denoted above images. Images show max intensity projection of confocal z-stack slices taken with 0.5 µm steps. c , Time-lapse imaging of mitotic microtubule dynamics TMR-star labeling of TUA5. Images taken every 30 sec, elapsed time (min) is shown. d , pUBQ10:SNAP-TUA5 and Col-0 (wildtype) seedlings were stained with 500 nM SNAP-Cell TMR-Star for 3h, lysed and analyzed by SDS-PAGE. Left panel: fluorescence; right panel Coomassie blue staining. e , Confocal images of mitotic cells in Arabidopsis root epidermis stained with TMR-Star. Spindles and phragmoplasts were observed. f , Root tip of Arabidopsis coexpressing p35S:YFP-LTI6b, p35:H2B-RFP, and pUBQ10:SNAP-TUA5. 3-day-old seedlings were incubated in 1/2MS containing 500 nM SNAP-Cell 647-SiR for 30 min. Scale bars: 10 µm. Experiments were repeated independently 3 times with comparable results.
    Figure Legend Snippet: SNAP-tag enabled in vivo imaging of tubulin in BY-2 cells and Arabidopsis a , Labeling mechanism of SNAP-tag. b , Cortical microtubules in SNAP-TUA5 expressing BY-2 cells with SNAP dyes denoted above images. Images show max intensity projection of confocal z-stack slices taken with 0.5 µm steps. c , Time-lapse imaging of mitotic microtubule dynamics TMR-star labeling of TUA5. Images taken every 30 sec, elapsed time (min) is shown. d , pUBQ10:SNAP-TUA5 and Col-0 (wildtype) seedlings were stained with 500 nM SNAP-Cell TMR-Star for 3h, lysed and analyzed by SDS-PAGE. Left panel: fluorescence; right panel Coomassie blue staining. e , Confocal images of mitotic cells in Arabidopsis root epidermis stained with TMR-Star. Spindles and phragmoplasts were observed. f , Root tip of Arabidopsis coexpressing p35S:YFP-LTI6b, p35:H2B-RFP, and pUBQ10:SNAP-TUA5. 3-day-old seedlings were incubated in 1/2MS containing 500 nM SNAP-Cell 647-SiR for 30 min. Scale bars: 10 µm. Experiments were repeated independently 3 times with comparable results.

    Techniques Used: In Vivo Imaging, Labeling, Expressing, Imaging, Staining, SDS Page, Fluorescence, Incubation

    15) Product Images from "Dual Bioorthogonal Labeling of the Amyloid-β Protein Precursor Facilitates Simultaneous Visualization of the Protein and Its Cleavage Products"

    Article Title: Dual Bioorthogonal Labeling of the Amyloid-β Protein Precursor Facilitates Simultaneous Visualization of the Protein and Its Cleavage Products

    Journal: Journal of Alzheimer's Disease

    doi: 10.3233/JAD-190898

    A, B) Time series of dual labelled AβPP-SNAP protein over a time frame of two minutes shows vesicular movement. HEK293T cells were transfected with the amber codon machinery and AβPP H13 > amber. Live cells were labeled with SNAP-Cell® 647-SiR (red) and mT-BDP-FL, which reacts with the ncAA (green). Labeled cells were imaged simultaneous by confocal microscopy (63× magnification). Blue arrows indicate nuclei, pink arrows indicate subcellular locations, where most AβPP is located.
    Figure Legend Snippet: A, B) Time series of dual labelled AβPP-SNAP protein over a time frame of two minutes shows vesicular movement. HEK293T cells were transfected with the amber codon machinery and AβPP H13 > amber. Live cells were labeled with SNAP-Cell® 647-SiR (red) and mT-BDP-FL, which reacts with the ncAA (green). Labeled cells were imaged simultaneous by confocal microscopy (63× magnification). Blue arrows indicate nuclei, pink arrows indicate subcellular locations, where most AβPP is located.

    Techniques Used: Transfection, Labeling, Confocal Microscopy

    16) Product Images from "BLOC-1 is required for selective membrane protein trafficking from endosomes to primary cilia"

    Article Title: BLOC-1 is required for selective membrane protein trafficking from endosomes to primary cilia

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201611138

    Overexpression of MyoVb perturbs polycytin-2 trafficking to the primary cilium. (A) Selected trafficking images of polycystin-2-GFP-SNAP IMCD Flp-In cells overexpressing Flag-MyoVb C-terminal tail. GFP and Arl13b antibody staining (green), SNAP TMR STAR (red), and Flag-MyoVb (blue). Insets depict newly synthesized polycystin-2-GFP-SNAP trafficking to the cilium in control or Flag-MyoVb-overexpressing cells. Insets also show accumulation of newly synthesized polycystin-2-GFP-SNAP in the recycling endosome in the Flag-MyoVb-overexpressing cells. Insets are 200% enlargements of the cilia and 170% enlargements of the recycling endosome. Bars, 10 µm. (B) Mean cilia length of polycystin-2-GFP-SNAP IMCD Flp-In control and Flag-MyoVb-overexpressing cells after the Golgi release. Cilia are shorter in Flag-MyoVb-overexpressing cells. (C) Mean SNAP ciliary fluorescence of Polycystin-2-GFP-SNAP delivery to the cilium after Golgi release. Polycystin-2 ciliary trafficking decreased in Flag-MyoVb-overexpressing cells. (D) Linear regression slope values of newly synthesized polycystin-2-GFP-SNAP in IMCD Flp-In control and Flag-MyoVb-overexpressing cells after the Golgi release using data points taken at 0, 1, 2, 4, and 6 h. Mean SNAP ciliary fluorescence and mean ciliary length were plotted from three independent experiments in which 30 cilia were quantified for each condition (control and Flag-MyoVb) at each time point ( n = 90 total cilia per time point). Error bars represent SEM. Data were analyzed using the unpaired Student’s t test. *, P
    Figure Legend Snippet: Overexpression of MyoVb perturbs polycytin-2 trafficking to the primary cilium. (A) Selected trafficking images of polycystin-2-GFP-SNAP IMCD Flp-In cells overexpressing Flag-MyoVb C-terminal tail. GFP and Arl13b antibody staining (green), SNAP TMR STAR (red), and Flag-MyoVb (blue). Insets depict newly synthesized polycystin-2-GFP-SNAP trafficking to the cilium in control or Flag-MyoVb-overexpressing cells. Insets also show accumulation of newly synthesized polycystin-2-GFP-SNAP in the recycling endosome in the Flag-MyoVb-overexpressing cells. Insets are 200% enlargements of the cilia and 170% enlargements of the recycling endosome. Bars, 10 µm. (B) Mean cilia length of polycystin-2-GFP-SNAP IMCD Flp-In control and Flag-MyoVb-overexpressing cells after the Golgi release. Cilia are shorter in Flag-MyoVb-overexpressing cells. (C) Mean SNAP ciliary fluorescence of Polycystin-2-GFP-SNAP delivery to the cilium after Golgi release. Polycystin-2 ciliary trafficking decreased in Flag-MyoVb-overexpressing cells. (D) Linear regression slope values of newly synthesized polycystin-2-GFP-SNAP in IMCD Flp-In control and Flag-MyoVb-overexpressing cells after the Golgi release using data points taken at 0, 1, 2, 4, and 6 h. Mean SNAP ciliary fluorescence and mean ciliary length were plotted from three independent experiments in which 30 cilia were quantified for each condition (control and Flag-MyoVb) at each time point ( n = 90 total cilia per time point). Error bars represent SEM. Data were analyzed using the unpaired Student’s t test. *, P

    Techniques Used: Over Expression, Staining, Synthesized, Fluorescence

    17) Product Images from "Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3"

    Article Title: Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3

    Journal: Journal of Virology

    doi: 10.1128/JVI.00477-18

    (A) Cell lysates from stable CHIKV cells. Live cells were stained with BG-TMR-Star and lysed with Glasgow lysis buffer. The lysate was then bound to plastic chamber slides overnight and imaged the following day. Images were acquired with an LSM880 microscope operated in Fast Airyscan mode. Cell lysates from uninfected, naive HuH-7 cells are shown as a control. (B) Zoomed-in views of the sample shown in panel A, providing higher-magnification views of nsP3-containing granules and corresponding bright-field images (transmission). (C) Cell lysates from HuH-7 cells infected with CHIKV ZsGreen-P3 . Samples were prepared as described for panel A and imaged in the green channel. (D) Schematic overview of different populations (P1 to P4) obtained during culture of stable CHIKV cells. (E) Wide-field microscopy of stable CHIKV cells passaged for 1 week in the absence of puromycin (P2). Naive HuH-7 cells and cells treated for 1 week with puromycin (P1) are shown as controls. Images were obtained with an IncuCyte Zoom live-cell imaging system. (F) Effect of puromycin treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. Confluence of the two populations was determined from images taken with an IncuCyte Zoom live-cell imaging system. (G) Effect of sodium arsenite treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. To induce cellular stress granules, sodium arsenite was added for at least 30 min. Cells were fixed and then stained for SNAP-nsP3 (cyan) and G3BP2 (magenta). Stained cells were imaged by Airyscan microscopy. FOVs 1 and 3 were centered on cells expressing ZsGreen, whereas FOVs 2 and 4 focused on cells that were ZsGreen negative.
    Figure Legend Snippet: (A) Cell lysates from stable CHIKV cells. Live cells were stained with BG-TMR-Star and lysed with Glasgow lysis buffer. The lysate was then bound to plastic chamber slides overnight and imaged the following day. Images were acquired with an LSM880 microscope operated in Fast Airyscan mode. Cell lysates from uninfected, naive HuH-7 cells are shown as a control. (B) Zoomed-in views of the sample shown in panel A, providing higher-magnification views of nsP3-containing granules and corresponding bright-field images (transmission). (C) Cell lysates from HuH-7 cells infected with CHIKV ZsGreen-P3 . Samples were prepared as described for panel A and imaged in the green channel. (D) Schematic overview of different populations (P1 to P4) obtained during culture of stable CHIKV cells. (E) Wide-field microscopy of stable CHIKV cells passaged for 1 week in the absence of puromycin (P2). Naive HuH-7 cells and cells treated for 1 week with puromycin (P1) are shown as controls. Images were obtained with an IncuCyte Zoom live-cell imaging system. (F) Effect of puromycin treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. Confluence of the two populations was determined from images taken with an IncuCyte Zoom live-cell imaging system. (G) Effect of sodium arsenite treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. To induce cellular stress granules, sodium arsenite was added for at least 30 min. Cells were fixed and then stained for SNAP-nsP3 (cyan) and G3BP2 (magenta). Stained cells were imaged by Airyscan microscopy. FOVs 1 and 3 were centered on cells expressing ZsGreen, whereas FOVs 2 and 4 focused on cells that were ZsGreen negative.

    Techniques Used: Staining, Lysis, Microscopy, Transmission Assay, Infection, Live Cell Imaging, Expressing

    nsP3 has a granular distribution in stable CHIKV cells and infected HuH-7 cells. (A) Schematic representation of tagged reporter viruses and noncytotoxic replicon encoding SNAP-nsP3. SGP, subgenomic promoter; PAC, puromycin- N -acetyltransferase; 2A, foot-and-mouth disease virus (FMDV) 2A autoprotease. (B) Subdiffraction confocal microscopy of BG-647-SiR-labeled stable CHIKV cells, imaged in the far-red channel. Cells were chemically fixed and stained with fluorescent BG-647-SiR, which irreversibly binds SNAP-tagged proteins. (C to E) Naive HuH-7 cells were infected with viral stocks of CHIKV SNAP-P3 , CHIKV ZsGreen-P3 , or CHIKV WT . For cells infected with CHIKV ZsGreen-P3 , images were acquired in the green channel. Cells infected with CHIKV WT were immunostained with anti-nsP3 antibodies to visualize untagged nsP3. Data shown are maximum-intensity projections of Z-stacks acquired on an Airyscan confocal system, operated in the superresolution mode. To enhance the appearance of dim structures, Icy software ( 84 ) was used to pseudocolor image channels with the predefined Fire lookup table based on pixel intensity. Color bars indicate the relative range of pixel intensity (white = high, purple = low, from 0 arbitrary units to 1). Nuclear counterstain (gray) was overlaid as a reference. Images displayed in the Fire view, based on a logarithmic scale, illustrate both high-intensity and low-intensity granules in the same image. (F) Size of nsP3-containing granules. Diameters of granules were extracted from the maximum-intensity projections of a total of 10 fields of view (FOVs; including panels B to E and five additional FOVs). Feret diameters represent the maximum distance between any two points of the extracted surface. (G) Airyscan microscopy of rod-containing cells infected with CHIKV SNAP-P3 . Zoomed-in views of nsP3-containing rods are provided in insets. (H) Mouse myoblast cell line (C2C12) replicating the SNAP-tagged noncytotoxic replicon. At 10 days after transfection and puromycin selection, cells were fixed and stained for SNAP-nsP3 with BG-TMR-Star. The ZsGreen channel is presented in grayscale view.
    Figure Legend Snippet: nsP3 has a granular distribution in stable CHIKV cells and infected HuH-7 cells. (A) Schematic representation of tagged reporter viruses and noncytotoxic replicon encoding SNAP-nsP3. SGP, subgenomic promoter; PAC, puromycin- N -acetyltransferase; 2A, foot-and-mouth disease virus (FMDV) 2A autoprotease. (B) Subdiffraction confocal microscopy of BG-647-SiR-labeled stable CHIKV cells, imaged in the far-red channel. Cells were chemically fixed and stained with fluorescent BG-647-SiR, which irreversibly binds SNAP-tagged proteins. (C to E) Naive HuH-7 cells were infected with viral stocks of CHIKV SNAP-P3 , CHIKV ZsGreen-P3 , or CHIKV WT . For cells infected with CHIKV ZsGreen-P3 , images were acquired in the green channel. Cells infected with CHIKV WT were immunostained with anti-nsP3 antibodies to visualize untagged nsP3. Data shown are maximum-intensity projections of Z-stacks acquired on an Airyscan confocal system, operated in the superresolution mode. To enhance the appearance of dim structures, Icy software ( 84 ) was used to pseudocolor image channels with the predefined Fire lookup table based on pixel intensity. Color bars indicate the relative range of pixel intensity (white = high, purple = low, from 0 arbitrary units to 1). Nuclear counterstain (gray) was overlaid as a reference. Images displayed in the Fire view, based on a logarithmic scale, illustrate both high-intensity and low-intensity granules in the same image. (F) Size of nsP3-containing granules. Diameters of granules were extracted from the maximum-intensity projections of a total of 10 fields of view (FOVs; including panels B to E and five additional FOVs). Feret diameters represent the maximum distance between any two points of the extracted surface. (G) Airyscan microscopy of rod-containing cells infected with CHIKV SNAP-P3 . Zoomed-in views of nsP3-containing rods are provided in insets. (H) Mouse myoblast cell line (C2C12) replicating the SNAP-tagged noncytotoxic replicon. At 10 days after transfection and puromycin selection, cells were fixed and stained for SNAP-nsP3 with BG-TMR-Star. The ZsGreen channel is presented in grayscale view.

    Techniques Used: Infection, Confocal Microscopy, Labeling, Staining, Software, Microscopy, Transfection, Selection

    18) Product Images from "Molecular coordination of Staphylococcus aureus cell division"

    Article Title: Molecular coordination of Staphylococcus aureus cell division

    Journal: eLife

    doi: 10.7554/eLife.32057

    STORM and SIM data. ( a ) EzrA-GFP (i) and SNAP-Cell TMR-Star labelled EzrA-SNAP (ii) localisation in SH4640 ( ezrA-gfp ΔezrA ) and SH4642 ( ezrA-snap ΔezrA ) by 3D-SIM, respectively. The images are maximum intensity projections of reconstructed z stacks. Scale bars 1 μm. 3D surface profiles of the circled area show distribution of fluorescence intensity of EzrA-GFP and EzrA-SNAP TMR-Star rings. ( b ) Localisation microscopy of EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ).
    Figure Legend Snippet: STORM and SIM data. ( a ) EzrA-GFP (i) and SNAP-Cell TMR-Star labelled EzrA-SNAP (ii) localisation in SH4640 ( ezrA-gfp ΔezrA ) and SH4642 ( ezrA-snap ΔezrA ) by 3D-SIM, respectively. The images are maximum intensity projections of reconstructed z stacks. Scale bars 1 μm. 3D surface profiles of the circled area show distribution of fluorescence intensity of EzrA-GFP and EzrA-SNAP TMR-Star rings. ( b ) Localisation microscopy of EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ).

    Techniques Used: Fluorescence, Microscopy

    Effect of FtsZ inhibitor PC190723 on S. aureus . ( a ) SH4652 ( ezrA-eyfp ΔezrA pCQ11-FtsZ-SNAP) grown in the presence of 50 μM IPTG in the absence (control) or presence of PC190723 (10 μg ml −1 ) for 0, 15, 30 and 60 min, labelled with SNAP-Cell TMR-Star was incubated with HADA for 5 min. Images are average intensity projections of z stacks. Scale bars 3 µm. Arrows indicate localisation defects. ( b ) Cell volume of S. aureus SH1000 during treatment with PC190723 (10 μg ml −1 ). Data are expressed as mean ±standard deviation.
    Figure Legend Snippet: Effect of FtsZ inhibitor PC190723 on S. aureus . ( a ) SH4652 ( ezrA-eyfp ΔezrA pCQ11-FtsZ-SNAP) grown in the presence of 50 μM IPTG in the absence (control) or presence of PC190723 (10 μg ml −1 ) for 0, 15, 30 and 60 min, labelled with SNAP-Cell TMR-Star was incubated with HADA for 5 min. Images are average intensity projections of z stacks. Scale bars 3 µm. Arrows indicate localisation defects. ( b ) Cell volume of S. aureus SH1000 during treatment with PC190723 (10 μg ml −1 ). Data are expressed as mean ±standard deviation.

    Techniques Used: Incubation, Standard Deviation

    EzrA fusions are functional. ( a ) Construction of S. aureus strains in which the only copy of ezrA is tagged (FL). Integration of pKASBAR-EzrA-FL at S. aureus lipase ( geh ) resulted in an ectopic copy of ezrA-fl under the control of the native ezrA promoter (P). A double-crossover event of pOB- ΔezrA allowed for marked with a tetracycline resistance (tetR) gene deletion of ezrA from its native chromosomal location. FL represents either eYFP, meYFP, GFP or SNAP. Not to scale. ( b ) Growth rates of ezrA fusions. EzrA-eYFP, EzrA-meYFP, EzrA-GFP and EzrA-SNAP complement native ezrA knock-out in SH4388 ( ezrA-eyfp ΔezrA ), SH4604 ( ezrA-meyfp ΔezrA ), SH4640 ( ezrA-gfp ΔezrA ) and SH4642 ( ezrA-snap ΔezrA ), respectively. The mutant strains (doubling time 24 min) showed similar growth to the wild type strain, SH1000 (doubling time 25 min). Growth rates were obtained by fitting an exponential growth equation to the most linear region of growth curves (R 2 > 0.98). Bacterial cultures were prepared in triplicate and the error bars represent standard deviation from the mean. ( c ) Epifluorescence microscopy images of EzrA-eYFP in SH4388 ( ezrA-eyfp ΔezrA ), EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ), EzrA-GFP in SH4640 ( ezrA-gfp ΔezrA ) and SNAP-Cell TMR-Star labelled EzrA-SNAP in SH4642 ( ezrA-snap ΔezrA ). Images are maximum intensity fluorescence projections of z stacks. Scale bars 3 μm. ( d ) EzrA-eYFP in SH4388 ( ezrA-eyfp ΔezrA ) and EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ) were detected by western blot analysis of total protein extracts using anti-GFP antibodies. Whole cell lysate of SH1000 and a recombinant GFP-HisTag protein were used as controls. Bands detected at ~95 kDa (EzrA-eYFP and EzrA-meYFP) and ~28 kDa (GFP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa. ( e ) EzrA-GFP in SH4640 ( ezrA-gfp ΔezrA ) was detected by immunoblot analysis of total protein extract using anti-GFP antibodies. Whole cell lysate of SH1000 and a recombinant GFP-HisTag protein were used as controls. Bands detected at ~95 kDa (EzrA-GFP) and ~28 kDa (GFP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa. ( f ) Whole cell lysate of SNAP-Cell TMR-Star labelled SH4642 ( ezrA-snap ΔezrA ) was resolved by 10% (w/v) SDS-PAGE and visualised by fluorescence detection. Whole cell lysate of SNAP-Cell TMR-Star labelled SH1000 and a purified SNAP-Cell TMR-Star labelled HisTag-SNAP protein were used as controls. Bands detected at ~85 kDa (EzrA-SNAP) and ~23 kDa (SNAP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa.
    Figure Legend Snippet: EzrA fusions are functional. ( a ) Construction of S. aureus strains in which the only copy of ezrA is tagged (FL). Integration of pKASBAR-EzrA-FL at S. aureus lipase ( geh ) resulted in an ectopic copy of ezrA-fl under the control of the native ezrA promoter (P). A double-crossover event of pOB- ΔezrA allowed for marked with a tetracycline resistance (tetR) gene deletion of ezrA from its native chromosomal location. FL represents either eYFP, meYFP, GFP or SNAP. Not to scale. ( b ) Growth rates of ezrA fusions. EzrA-eYFP, EzrA-meYFP, EzrA-GFP and EzrA-SNAP complement native ezrA knock-out in SH4388 ( ezrA-eyfp ΔezrA ), SH4604 ( ezrA-meyfp ΔezrA ), SH4640 ( ezrA-gfp ΔezrA ) and SH4642 ( ezrA-snap ΔezrA ), respectively. The mutant strains (doubling time 24 min) showed similar growth to the wild type strain, SH1000 (doubling time 25 min). Growth rates were obtained by fitting an exponential growth equation to the most linear region of growth curves (R 2 > 0.98). Bacterial cultures were prepared in triplicate and the error bars represent standard deviation from the mean. ( c ) Epifluorescence microscopy images of EzrA-eYFP in SH4388 ( ezrA-eyfp ΔezrA ), EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ), EzrA-GFP in SH4640 ( ezrA-gfp ΔezrA ) and SNAP-Cell TMR-Star labelled EzrA-SNAP in SH4642 ( ezrA-snap ΔezrA ). Images are maximum intensity fluorescence projections of z stacks. Scale bars 3 μm. ( d ) EzrA-eYFP in SH4388 ( ezrA-eyfp ΔezrA ) and EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ) were detected by western blot analysis of total protein extracts using anti-GFP antibodies. Whole cell lysate of SH1000 and a recombinant GFP-HisTag protein were used as controls. Bands detected at ~95 kDa (EzrA-eYFP and EzrA-meYFP) and ~28 kDa (GFP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa. ( e ) EzrA-GFP in SH4640 ( ezrA-gfp ΔezrA ) was detected by immunoblot analysis of total protein extract using anti-GFP antibodies. Whole cell lysate of SH1000 and a recombinant GFP-HisTag protein were used as controls. Bands detected at ~95 kDa (EzrA-GFP) and ~28 kDa (GFP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa. ( f ) Whole cell lysate of SNAP-Cell TMR-Star labelled SH4642 ( ezrA-snap ΔezrA ) was resolved by 10% (w/v) SDS-PAGE and visualised by fluorescence detection. Whole cell lysate of SNAP-Cell TMR-Star labelled SH1000 and a purified SNAP-Cell TMR-Star labelled HisTag-SNAP protein were used as controls. Bands detected at ~85 kDa (EzrA-SNAP) and ~23 kDa (SNAP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa.

    Techniques Used: Functional Assay, Knock-Out, Mutagenesis, Standard Deviation, Epifluorescence Microscopy, Fluorescence, Western Blot, Recombinant, SDS Page, Purification

    19) Product Images from "POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication"

    Article Title: POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2018.08.043

    Transient Depletion of the POLE3-POLE4 Complex Affects Histone Deposition in SNAP Tag H3.1-Expressing Cells (A) In HeLa H3.1-SNAP: at left, quench-chase-pulse experiment to follow new H3.1; at right, pulse-chase experiment to follow parental H3.1. A quenching step labels all pre-existing histones with a non-fluorescent dye. A chase step allows synthesis and deposition of new unlabeled H3.1-SNAP. A pulse using the fluorophore TMR (red) labels available H3.1-SNAP. EdU incorporation at the end of the assay allows the detection of replicated DNA (green). A Triton extraction step is performed prior to fixation to eliminate soluble histones and analyze chromatin-bound H3.1. (B) Representative images of new (left) or parental (right) H3.1 (TMR, red) and replication sites (EdU, green) in control, POLE1-depleted, POLE3-depleted, or POLE4-depleted conditions. Scale bars, 10 μm. (C) Quantification of TMR fluorescence signal per nucleus normalized to control mean for new (left) or parental (right) H3.1 in control, POLE1-depleted, POLE3-depleted, or POLE4-depleted conditions (n = 3). For new H3.1 (left), only cells in S phase were quantified.
    Figure Legend Snippet: Transient Depletion of the POLE3-POLE4 Complex Affects Histone Deposition in SNAP Tag H3.1-Expressing Cells (A) In HeLa H3.1-SNAP: at left, quench-chase-pulse experiment to follow new H3.1; at right, pulse-chase experiment to follow parental H3.1. A quenching step labels all pre-existing histones with a non-fluorescent dye. A chase step allows synthesis and deposition of new unlabeled H3.1-SNAP. A pulse using the fluorophore TMR (red) labels available H3.1-SNAP. EdU incorporation at the end of the assay allows the detection of replicated DNA (green). A Triton extraction step is performed prior to fixation to eliminate soluble histones and analyze chromatin-bound H3.1. (B) Representative images of new (left) or parental (right) H3.1 (TMR, red) and replication sites (EdU, green) in control, POLE1-depleted, POLE3-depleted, or POLE4-depleted conditions. Scale bars, 10 μm. (C) Quantification of TMR fluorescence signal per nucleus normalized to control mean for new (left) or parental (right) H3.1 in control, POLE1-depleted, POLE3-depleted, or POLE4-depleted conditions (n = 3). For new H3.1 (left), only cells in S phase were quantified.

    Techniques Used: Expressing, Pulse Chase, Fluorescence

    20) Product Images from "Rab11a-positive compartments in proximal tubule cells sort fluid-phase and membrane cargo"

    Article Title: Rab11a-positive compartments in proximal tubule cells sort fluid-phase and membrane cargo

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00236.2013

    Rab11a-positive compartments in PTCs are highly dynamic. PTCs cultured on MatTek dishes were transfected with cDNA encoding SNAP-tagged Rab11a. After 2 days, cells were incubated with Cell-SNAP TMR-Star to label the SNAP tag, washed, and then imaged by
    Figure Legend Snippet: Rab11a-positive compartments in PTCs are highly dynamic. PTCs cultured on MatTek dishes were transfected with cDNA encoding SNAP-tagged Rab11a. After 2 days, cells were incubated with Cell-SNAP TMR-Star to label the SNAP tag, washed, and then imaged by

    Techniques Used: Cell Culture, Transfection, Incubation

    21) Product Images from "Structural transitions of centromeric chromatin regulate the cell cycle-dependent recruitment of CENP-N"

    Article Title: Structural transitions of centromeric chromatin regulate the cell cycle-dependent recruitment of CENP-N

    Journal: Genes & Development

    doi: 10.1101/gad.259432.115

    Specific loading of CENP-N in middle/late S phase (see also Supplemental Fig. S6). ( A ) Representative images of HeLa cells expressing SNAP-CENP-N in different cell cycle phases. SNAP-CENP-N was labeled with TMR-Star (red), and cell cycle phases were determined
    Figure Legend Snippet: Specific loading of CENP-N in middle/late S phase (see also Supplemental Fig. S6). ( A ) Representative images of HeLa cells expressing SNAP-CENP-N in different cell cycle phases. SNAP-CENP-N was labeled with TMR-Star (red), and cell cycle phases were determined

    Techniques Used: Expressing, Labeling

    22) Product Images from "Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3"

    Article Title: Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3

    Journal: Journal of Virology

    doi: 10.1128/JVI.00477-18

    (A) Cell lysates from stable CHIKV cells. Live cells were stained with BG-TMR-Star and lysed with Glasgow lysis buffer. The lysate was then bound to plastic chamber slides overnight and imaged the following day. Images were acquired with an LSM880 microscope operated in Fast Airyscan mode. Cell lysates from uninfected, naive HuH-7 cells are shown as a control. (B) Zoomed-in views of the sample shown in panel A, providing higher-magnification views of nsP3-containing granules and corresponding bright-field images (transmission). (C) Cell lysates from HuH-7 cells infected with CHIKV ZsGreen-P3 . Samples were prepared as described for panel A and imaged in the green channel. (D) Schematic overview of different populations (P1 to P4) obtained during culture of stable CHIKV cells. (E) Wide-field microscopy of stable CHIKV cells passaged for 1 week in the absence of puromycin (P2). Naive HuH-7 cells and cells treated for 1 week with puromycin (P1) are shown as controls. Images were obtained with an IncuCyte Zoom live-cell imaging system. (F) Effect of puromycin treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. Confluence of the two populations was determined from images taken with an IncuCyte Zoom live-cell imaging system. (G) Effect of sodium arsenite treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. To induce cellular stress granules, sodium arsenite was added for at least 30 min. Cells were fixed and then stained for SNAP-nsP3 (cyan) and G3BP2 (magenta). Stained cells were imaged by Airyscan microscopy. FOVs 1 and 3 were centered on cells expressing ZsGreen, whereas FOVs 2 and 4 focused on cells that were ZsGreen negative.
    Figure Legend Snippet: (A) Cell lysates from stable CHIKV cells. Live cells were stained with BG-TMR-Star and lysed with Glasgow lysis buffer. The lysate was then bound to plastic chamber slides overnight and imaged the following day. Images were acquired with an LSM880 microscope operated in Fast Airyscan mode. Cell lysates from uninfected, naive HuH-7 cells are shown as a control. (B) Zoomed-in views of the sample shown in panel A, providing higher-magnification views of nsP3-containing granules and corresponding bright-field images (transmission). (C) Cell lysates from HuH-7 cells infected with CHIKV ZsGreen-P3 . Samples were prepared as described for panel A and imaged in the green channel. (D) Schematic overview of different populations (P1 to P4) obtained during culture of stable CHIKV cells. (E) Wide-field microscopy of stable CHIKV cells passaged for 1 week in the absence of puromycin (P2). Naive HuH-7 cells and cells treated for 1 week with puromycin (P1) are shown as controls. Images were obtained with an IncuCyte Zoom live-cell imaging system. (F) Effect of puromycin treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. Confluence of the two populations was determined from images taken with an IncuCyte Zoom live-cell imaging system. (G) Effect of sodium arsenite treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. To induce cellular stress granules, sodium arsenite was added for at least 30 min. Cells were fixed and then stained for SNAP-nsP3 (cyan) and G3BP2 (magenta). Stained cells were imaged by Airyscan microscopy. FOVs 1 and 3 were centered on cells expressing ZsGreen, whereas FOVs 2 and 4 focused on cells that were ZsGreen negative.

    Techniques Used: Staining, Lysis, Microscopy, Transmission Assay, Infection, Live Cell Imaging, Expressing

    23) Product Images from "Molecular coordination of Staphylococcus aureus cell division"

    Article Title: Molecular coordination of Staphylococcus aureus cell division

    Journal: eLife

    doi: 10.7554/eLife.32057

    STORM and SIM data. ( a ) EzrA-GFP (i) and SNAP-Cell TMR-Star labelled EzrA-SNAP (ii) localisation in SH4640 ( ezrA-gfp ΔezrA ) and SH4642 ( ezrA-snap ΔezrA ) by 3D-SIM, respectively. The images are maximum intensity projections of reconstructed z stacks. Scale bars 1 μm. 3D surface profiles of the circled area show distribution of fluorescence intensity of EzrA-GFP and EzrA-SNAP TMR-Star rings. ( b ) Localisation microscopy of EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ).
    Figure Legend Snippet: STORM and SIM data. ( a ) EzrA-GFP (i) and SNAP-Cell TMR-Star labelled EzrA-SNAP (ii) localisation in SH4640 ( ezrA-gfp ΔezrA ) and SH4642 ( ezrA-snap ΔezrA ) by 3D-SIM, respectively. The images are maximum intensity projections of reconstructed z stacks. Scale bars 1 μm. 3D surface profiles of the circled area show distribution of fluorescence intensity of EzrA-GFP and EzrA-SNAP TMR-Star rings. ( b ) Localisation microscopy of EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ).

    Techniques Used: Fluorescence, Microscopy

    Effect of FtsZ inhibitor PC190723 on S. aureus . ( a ) SH4652 ( ezrA-eyfp ΔezrA pCQ11-FtsZ-SNAP) grown in the presence of 50 μM IPTG in the absence (control) or presence of PC190723 (10 μg ml −1 ) for 0, 15, 30 and 60 min, labelled with SNAP-Cell TMR-Star was incubated with HADA for 5 min. Images are average intensity projections of z stacks. Scale bars 3 µm. Arrows indicate localisation defects. ( b ) Cell volume of S. aureus SH1000 during treatment with PC190723 (10 μg ml −1 ). Data are expressed as mean ±standard deviation.
    Figure Legend Snippet: Effect of FtsZ inhibitor PC190723 on S. aureus . ( a ) SH4652 ( ezrA-eyfp ΔezrA pCQ11-FtsZ-SNAP) grown in the presence of 50 μM IPTG in the absence (control) or presence of PC190723 (10 μg ml −1 ) for 0, 15, 30 and 60 min, labelled with SNAP-Cell TMR-Star was incubated with HADA for 5 min. Images are average intensity projections of z stacks. Scale bars 3 µm. Arrows indicate localisation defects. ( b ) Cell volume of S. aureus SH1000 during treatment with PC190723 (10 μg ml −1 ). Data are expressed as mean ±standard deviation.

    Techniques Used: Incubation, Standard Deviation

    EzrA fusions are functional. ( a ) Construction of S. aureus strains in which the only copy of ezrA is tagged (FL). Integration of pKASBAR-EzrA-FL at S. aureus lipase ( geh ) resulted in an ectopic copy of ezrA-fl under the control of the native ezrA promoter (P). A double-crossover event of pOB- ΔezrA allowed for marked with a tetracycline resistance (tetR) gene deletion of ezrA from its native chromosomal location. FL represents either eYFP, meYFP, GFP or SNAP. Not to scale. ( b ) Growth rates of ezrA fusions. EzrA-eYFP, EzrA-meYFP, EzrA-GFP and EzrA-SNAP complement native ezrA knock-out in SH4388 ( ezrA-eyfp ΔezrA ), SH4604 ( ezrA-meyfp ΔezrA ), SH4640 ( ezrA-gfp ΔezrA ) and SH4642 ( ezrA-snap ΔezrA ), respectively. The mutant strains (doubling time 24 min) showed similar growth to the wild type strain, SH1000 (doubling time 25 min). Growth rates were obtained by fitting an exponential growth equation to the most linear region of growth curves (R 2 > 0.98). Bacterial cultures were prepared in triplicate and the error bars represent standard deviation from the mean. ( c ) Epifluorescence microscopy images of EzrA-eYFP in SH4388 ( ezrA-eyfp ΔezrA ), EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ), EzrA-GFP in SH4640 ( ezrA-gfp ΔezrA ) and SNAP-Cell TMR-Star labelled EzrA-SNAP in SH4642 ( ezrA-snap ΔezrA ). Images are maximum intensity fluorescence projections of z stacks. Scale bars 3 μm. ( d ) EzrA-eYFP in SH4388 ( ezrA-eyfp ΔezrA ) and EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ) were detected by western blot analysis of total protein extracts using anti-GFP antibodies. Whole cell lysate of SH1000 and a recombinant GFP-HisTag protein were used as controls. Bands detected at ~95 kDa (EzrA-eYFP and EzrA-meYFP) and ~28 kDa (GFP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa. ( e ) EzrA-GFP in SH4640 ( ezrA-gfp ΔezrA ) was detected by immunoblot analysis of total protein extract using anti-GFP antibodies. Whole cell lysate of SH1000 and a recombinant GFP-HisTag protein were used as controls. Bands detected at ~95 kDa (EzrA-GFP) and ~28 kDa (GFP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa. ( f ) Whole cell lysate of SNAP-Cell TMR-Star labelled SH4642 ( ezrA-snap ΔezrA ) was resolved by 10% (w/v) SDS-PAGE and visualised by fluorescence detection. Whole cell lysate of SNAP-Cell TMR-Star labelled SH1000 and a purified SNAP-Cell TMR-Star labelled HisTag-SNAP protein were used as controls. Bands detected at ~85 kDa (EzrA-SNAP) and ~23 kDa (SNAP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa.
    Figure Legend Snippet: EzrA fusions are functional. ( a ) Construction of S. aureus strains in which the only copy of ezrA is tagged (FL). Integration of pKASBAR-EzrA-FL at S. aureus lipase ( geh ) resulted in an ectopic copy of ezrA-fl under the control of the native ezrA promoter (P). A double-crossover event of pOB- ΔezrA allowed for marked with a tetracycline resistance (tetR) gene deletion of ezrA from its native chromosomal location. FL represents either eYFP, meYFP, GFP or SNAP. Not to scale. ( b ) Growth rates of ezrA fusions. EzrA-eYFP, EzrA-meYFP, EzrA-GFP and EzrA-SNAP complement native ezrA knock-out in SH4388 ( ezrA-eyfp ΔezrA ), SH4604 ( ezrA-meyfp ΔezrA ), SH4640 ( ezrA-gfp ΔezrA ) and SH4642 ( ezrA-snap ΔezrA ), respectively. The mutant strains (doubling time 24 min) showed similar growth to the wild type strain, SH1000 (doubling time 25 min). Growth rates were obtained by fitting an exponential growth equation to the most linear region of growth curves (R 2 > 0.98). Bacterial cultures were prepared in triplicate and the error bars represent standard deviation from the mean. ( c ) Epifluorescence microscopy images of EzrA-eYFP in SH4388 ( ezrA-eyfp ΔezrA ), EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ), EzrA-GFP in SH4640 ( ezrA-gfp ΔezrA ) and SNAP-Cell TMR-Star labelled EzrA-SNAP in SH4642 ( ezrA-snap ΔezrA ). Images are maximum intensity fluorescence projections of z stacks. Scale bars 3 μm. ( d ) EzrA-eYFP in SH4388 ( ezrA-eyfp ΔezrA ) and EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ) were detected by western blot analysis of total protein extracts using anti-GFP antibodies. Whole cell lysate of SH1000 and a recombinant GFP-HisTag protein were used as controls. Bands detected at ~95 kDa (EzrA-eYFP and EzrA-meYFP) and ~28 kDa (GFP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa. ( e ) EzrA-GFP in SH4640 ( ezrA-gfp ΔezrA ) was detected by immunoblot analysis of total protein extract using anti-GFP antibodies. Whole cell lysate of SH1000 and a recombinant GFP-HisTag protein were used as controls. Bands detected at ~95 kDa (EzrA-GFP) and ~28 kDa (GFP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa. ( f ) Whole cell lysate of SNAP-Cell TMR-Star labelled SH4642 ( ezrA-snap ΔezrA ) was resolved by 10% (w/v) SDS-PAGE and visualised by fluorescence detection. Whole cell lysate of SNAP-Cell TMR-Star labelled SH1000 and a purified SNAP-Cell TMR-Star labelled HisTag-SNAP protein were used as controls. Bands detected at ~85 kDa (EzrA-SNAP) and ~23 kDa (SNAP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa.

    Techniques Used: Functional Assay, Knock-Out, Mutagenesis, Standard Deviation, Epifluorescence Microscopy, Fluorescence, Western Blot, Recombinant, SDS Page, Purification

    24) Product Images from "Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings"

    Article Title: Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings

    Journal: bioRxiv

    doi: 10.1101/2020.03.09.983924

    SNAP-tag enabled in vivo imaging of tubulin in BY-2 cells and Arabidopsis a , Labeling mechanism of SNAP-tag. b , Cortical microtubules in SNAP-TUA5 expressing BY-2 cells with SNAP dyes denoted above images. Images show max intensity projection of confocal z-stack slices taken with 0.5 µm steps. c , Time-lapse imaging of mitotic microtubule dynamics TMR-star labeling of TUA5. Images taken every 30 sec, elapsed time (min) is shown. d , pUBQ10:SNAP-TUA5 and Col-0 (wildtype) seedlings were stained with 500 nM SNAP-Cell TMR-Star for 3h, lysed and analyzed by SDS-PAGE. Left panel: fluorescence; right panel Coomassie blue staining. e , Confocal images of mitotic cells in Arabidopsis root epidermis stained with TMR-Star. Spindles and phragmoplasts were observed. f , Root tip of Arabidopsis coexpressing p35S:YFP-LTI6b, p35:H2B-RFP, and pUBQ10:SNAP-TUA5. 3-day-old seedlings were incubated in 1/2MS containing 500 nM SNAP-Cell 647-SiR for 30 min. Scale bars: 10 µm. Experiments were repeated independently 3 times with comparable results.
    Figure Legend Snippet: SNAP-tag enabled in vivo imaging of tubulin in BY-2 cells and Arabidopsis a , Labeling mechanism of SNAP-tag. b , Cortical microtubules in SNAP-TUA5 expressing BY-2 cells with SNAP dyes denoted above images. Images show max intensity projection of confocal z-stack slices taken with 0.5 µm steps. c , Time-lapse imaging of mitotic microtubule dynamics TMR-star labeling of TUA5. Images taken every 30 sec, elapsed time (min) is shown. d , pUBQ10:SNAP-TUA5 and Col-0 (wildtype) seedlings were stained with 500 nM SNAP-Cell TMR-Star for 3h, lysed and analyzed by SDS-PAGE. Left panel: fluorescence; right panel Coomassie blue staining. e , Confocal images of mitotic cells in Arabidopsis root epidermis stained with TMR-Star. Spindles and phragmoplasts were observed. f , Root tip of Arabidopsis coexpressing p35S:YFP-LTI6b, p35:H2B-RFP, and pUBQ10:SNAP-TUA5. 3-day-old seedlings were incubated in 1/2MS containing 500 nM SNAP-Cell 647-SiR for 30 min. Scale bars: 10 µm. Experiments were repeated independently 3 times with comparable results.

    Techniques Used: In Vivo Imaging, Labeling, Expressing, Imaging, Staining, SDS Page, Fluorescence, Incubation

    25) Product Images from "SH2 domains serve as lipid binding modules for pTyr-signaling proteins"

    Article Title: SH2 domains serve as lipid binding modules for pTyr-signaling proteins

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2016.01.027

    Single molecule tracking of ZAP70 and TCR-ζ in the TCR complex. (A)-(B) Representative images of EGFP-ZAP70 WT (A) or K206E/K251E (B) and SNAP-TMR-labeled TCR-ζ in a P116 cell before and after OKT3 stimulation are shown. Green, red, and
    Figure Legend Snippet: Single molecule tracking of ZAP70 and TCR-ζ in the TCR complex. (A)-(B) Representative images of EGFP-ZAP70 WT (A) or K206E/K251E (B) and SNAP-TMR-labeled TCR-ζ in a P116 cell before and after OKT3 stimulation are shown. Green, red, and

    Techniques Used: Labeling

    26) Product Images from "Molecular coordination of Staphylococcus aureus cell division"

    Article Title: Molecular coordination of Staphylococcus aureus cell division

    Journal: eLife

    doi: 10.7554/eLife.32057

    STORM and SIM data. ( a ) EzrA-GFP (i) and SNAP-Cell TMR-Star labelled EzrA-SNAP (ii) localisation in SH4640 ( ezrA-gfp ΔezrA ) and SH4642 ( ezrA-snap ΔezrA ) by 3D-SIM, respectively. The images are maximum intensity projections of reconstructed z stacks. Scale bars 1 μm. 3D surface profiles of the circled area show distribution of fluorescence intensity of EzrA-GFP and EzrA-SNAP TMR-Star rings. ( b ) Localisation microscopy of EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ).
    Figure Legend Snippet: STORM and SIM data. ( a ) EzrA-GFP (i) and SNAP-Cell TMR-Star labelled EzrA-SNAP (ii) localisation in SH4640 ( ezrA-gfp ΔezrA ) and SH4642 ( ezrA-snap ΔezrA ) by 3D-SIM, respectively. The images are maximum intensity projections of reconstructed z stacks. Scale bars 1 μm. 3D surface profiles of the circled area show distribution of fluorescence intensity of EzrA-GFP and EzrA-SNAP TMR-Star rings. ( b ) Localisation microscopy of EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ).

    Techniques Used: Fluorescence, Microscopy

    Effect of FtsZ inhibitor PC190723 on S. aureus . ( a ) SH4652 ( ezrA-eyfp ΔezrA pCQ11-FtsZ-SNAP) grown in the presence of 50 μM IPTG in the absence (control) or presence of PC190723 (10 μg ml −1 ) for 0, 15, 30 and 60 min, labelled with SNAP-Cell TMR-Star was incubated with HADA for 5 min. Images are average intensity projections of z stacks. Scale bars 3 µm. Arrows indicate localisation defects. ( b ) Cell volume of S. aureus SH1000 during treatment with PC190723 (10 μg ml −1 ). Data are expressed as mean ±standard deviation.
    Figure Legend Snippet: Effect of FtsZ inhibitor PC190723 on S. aureus . ( a ) SH4652 ( ezrA-eyfp ΔezrA pCQ11-FtsZ-SNAP) grown in the presence of 50 μM IPTG in the absence (control) or presence of PC190723 (10 μg ml −1 ) for 0, 15, 30 and 60 min, labelled with SNAP-Cell TMR-Star was incubated with HADA for 5 min. Images are average intensity projections of z stacks. Scale bars 3 µm. Arrows indicate localisation defects. ( b ) Cell volume of S. aureus SH1000 during treatment with PC190723 (10 μg ml −1 ). Data are expressed as mean ±standard deviation.

    Techniques Used: Incubation, Standard Deviation

    EzrA fusions are functional. ( a ) Construction of S. aureus strains in which the only copy of ezrA is tagged (FL). Integration of pKASBAR-EzrA-FL at S. aureus lipase ( geh ) resulted in an ectopic copy of ezrA-fl under the control of the native ezrA promoter (P). A double-crossover event of pOB- ΔezrA allowed for marked with a tetracycline resistance (tetR) gene deletion of ezrA from its native chromosomal location. FL represents either eYFP, meYFP, GFP or SNAP. Not to scale. ( b ) Growth rates of ezrA fusions. EzrA-eYFP, EzrA-meYFP, EzrA-GFP and EzrA-SNAP complement native ezrA knock-out in SH4388 ( ezrA-eyfp ΔezrA ), SH4604 ( ezrA-meyfp ΔezrA ), SH4640 ( ezrA-gfp ΔezrA ) and SH4642 ( ezrA-snap ΔezrA ), respectively. The mutant strains (doubling time 24 min) showed similar growth to the wild type strain, SH1000 (doubling time 25 min). Growth rates were obtained by fitting an exponential growth equation to the most linear region of growth curves (R 2 > 0.98). Bacterial cultures were prepared in triplicate and the error bars represent standard deviation from the mean. ( c ) Epifluorescence microscopy images of EzrA-eYFP in SH4388 ( ezrA-eyfp ΔezrA ), EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ), EzrA-GFP in SH4640 ( ezrA-gfp ΔezrA ) and SNAP-Cell TMR-Star labelled EzrA-SNAP in SH4642 ( ezrA-snap ΔezrA ). Images are maximum intensity fluorescence projections of z stacks. Scale bars 3 μm. ( d ) EzrA-eYFP in SH4388 ( ezrA-eyfp ΔezrA ) and EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ) were detected by western blot analysis of total protein extracts using anti-GFP antibodies. Whole cell lysate of SH1000 and a recombinant GFP-HisTag protein were used as controls. Bands detected at ~95 kDa (EzrA-eYFP and EzrA-meYFP) and ~28 kDa (GFP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa. ( e ) EzrA-GFP in SH4640 ( ezrA-gfp ΔezrA ) was detected by immunoblot analysis of total protein extract using anti-GFP antibodies. Whole cell lysate of SH1000 and a recombinant GFP-HisTag protein were used as controls. Bands detected at ~95 kDa (EzrA-GFP) and ~28 kDa (GFP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa. ( f ) Whole cell lysate of SNAP-Cell TMR-Star labelled SH4642 ( ezrA-snap ΔezrA ) was resolved by 10% (w/v) SDS-PAGE and visualised by fluorescence detection. Whole cell lysate of SNAP-Cell TMR-Star labelled SH1000 and a purified SNAP-Cell TMR-Star labelled HisTag-SNAP protein were used as controls. Bands detected at ~85 kDa (EzrA-SNAP) and ~23 kDa (SNAP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa.
    Figure Legend Snippet: EzrA fusions are functional. ( a ) Construction of S. aureus strains in which the only copy of ezrA is tagged (FL). Integration of pKASBAR-EzrA-FL at S. aureus lipase ( geh ) resulted in an ectopic copy of ezrA-fl under the control of the native ezrA promoter (P). A double-crossover event of pOB- ΔezrA allowed for marked with a tetracycline resistance (tetR) gene deletion of ezrA from its native chromosomal location. FL represents either eYFP, meYFP, GFP or SNAP. Not to scale. ( b ) Growth rates of ezrA fusions. EzrA-eYFP, EzrA-meYFP, EzrA-GFP and EzrA-SNAP complement native ezrA knock-out in SH4388 ( ezrA-eyfp ΔezrA ), SH4604 ( ezrA-meyfp ΔezrA ), SH4640 ( ezrA-gfp ΔezrA ) and SH4642 ( ezrA-snap ΔezrA ), respectively. The mutant strains (doubling time 24 min) showed similar growth to the wild type strain, SH1000 (doubling time 25 min). Growth rates were obtained by fitting an exponential growth equation to the most linear region of growth curves (R 2 > 0.98). Bacterial cultures were prepared in triplicate and the error bars represent standard deviation from the mean. ( c ) Epifluorescence microscopy images of EzrA-eYFP in SH4388 ( ezrA-eyfp ΔezrA ), EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ), EzrA-GFP in SH4640 ( ezrA-gfp ΔezrA ) and SNAP-Cell TMR-Star labelled EzrA-SNAP in SH4642 ( ezrA-snap ΔezrA ). Images are maximum intensity fluorescence projections of z stacks. Scale bars 3 μm. ( d ) EzrA-eYFP in SH4388 ( ezrA-eyfp ΔezrA ) and EzrA-meYFP in SH4604 ( ezrA-meyfp ΔezrA ) were detected by western blot analysis of total protein extracts using anti-GFP antibodies. Whole cell lysate of SH1000 and a recombinant GFP-HisTag protein were used as controls. Bands detected at ~95 kDa (EzrA-eYFP and EzrA-meYFP) and ~28 kDa (GFP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa. ( e ) EzrA-GFP in SH4640 ( ezrA-gfp ΔezrA ) was detected by immunoblot analysis of total protein extract using anti-GFP antibodies. Whole cell lysate of SH1000 and a recombinant GFP-HisTag protein were used as controls. Bands detected at ~95 kDa (EzrA-GFP) and ~28 kDa (GFP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa. ( f ) Whole cell lysate of SNAP-Cell TMR-Star labelled SH4642 ( ezrA-snap ΔezrA ) was resolved by 10% (w/v) SDS-PAGE and visualised by fluorescence detection. Whole cell lysate of SNAP-Cell TMR-Star labelled SH1000 and a purified SNAP-Cell TMR-Star labelled HisTag-SNAP protein were used as controls. Bands detected at ~85 kDa (EzrA-SNAP) and ~23 kDa (SNAP-HisTag) are indicated with black arrows. Sizes of a protein ladder are shown in kDa.

    Techniques Used: Functional Assay, Knock-Out, Mutagenesis, Standard Deviation, Epifluorescence Microscopy, Fluorescence, Western Blot, Recombinant, SDS Page, Purification

    27) Product Images from "Genome-Wide Analysis of Cell Type-Specific Gene Transcription during Spore Formation in Clostridium difficileThe Spore Differentiation Pathway in the Enteric Pathogen Clostridium difficile"

    Article Title: Genome-Wide Analysis of Cell Type-Specific Gene Transcription during Spore Formation in Clostridium difficileThe Spore Differentiation Pathway in the Enteric Pathogen Clostridium difficile

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003756

    Fluorescence of a P spoIIR -SNAP fusion in strain 630Δerm and in a spo0A , sigF or sigE mutant. Cells of the C. difficile 630Δerm strain, and of the spo0A , sigF and sigE mutants carrying a P spoIIR -SNAP Cd transcriptional fusion in a multicopy plasmid were collected 24 h of following inocculation in SM broth. Cells were labelled with the fluorescent substrate TMR to allow localization of SNAP Cd production driven by the spoIIR promoter, stained with the DNA marker DAPI and the membrane dye MTG and examined by phase contrast and fluorescence microscopy.
    Figure Legend Snippet: Fluorescence of a P spoIIR -SNAP fusion in strain 630Δerm and in a spo0A , sigF or sigE mutant. Cells of the C. difficile 630Δerm strain, and of the spo0A , sigF and sigE mutants carrying a P spoIIR -SNAP Cd transcriptional fusion in a multicopy plasmid were collected 24 h of following inocculation in SM broth. Cells were labelled with the fluorescent substrate TMR to allow localization of SNAP Cd production driven by the spoIIR promoter, stained with the DNA marker DAPI and the membrane dye MTG and examined by phase contrast and fluorescence microscopy.

    Techniques Used: Fluorescence, Mutagenesis, Plasmid Preparation, Staining, Marker, Microscopy

    28) Product Images from "Segregation of molecules at cell division reveals native protein localization"

    Article Title: Segregation of molecules at cell division reveals native protein localization

    Journal: Nature methods

    doi: 10.1038/nmeth.1955

    ( a ) Immunofluorescence microscopy of ClpX in wildtype (left), ClpX-Venus YFP (middle) and Δ clpX (right) strains. Insets are phase images and a close-up is shown for the wildtype. ( b ) Fluorescence images show bacteria expressing the ClpP-SNAP tag labeled with TMR (tetramethylrhodamine), compared to wildtype (right). Insets show phase images. ( c ) Cartoons of a fluorescent protein (yellow) forming a weak anti-parallel dimer and of avidity effects potentially clustering tagged ClpX hexamers (blue). ( d ) Fluorescence images of bacteria expressing the indicated constructs. The cell outline (red) is shown for cells with weak cytoplasmic signal. ( e,f ) HILO microscopy of gently fixed cells with ClpX-mGFPmut3 ( e ) and ClpP-mGFPmut3 (f). ( g ) Live-cell HILO microscopy of cells expressing ClpP-mGFPmut3. Scale bars,1 μm.
    Figure Legend Snippet: ( a ) Immunofluorescence microscopy of ClpX in wildtype (left), ClpX-Venus YFP (middle) and Δ clpX (right) strains. Insets are phase images and a close-up is shown for the wildtype. ( b ) Fluorescence images show bacteria expressing the ClpP-SNAP tag labeled with TMR (tetramethylrhodamine), compared to wildtype (right). Insets show phase images. ( c ) Cartoons of a fluorescent protein (yellow) forming a weak anti-parallel dimer and of avidity effects potentially clustering tagged ClpX hexamers (blue). ( d ) Fluorescence images of bacteria expressing the indicated constructs. The cell outline (red) is shown for cells with weak cytoplasmic signal. ( e,f ) HILO microscopy of gently fixed cells with ClpX-mGFPmut3 ( e ) and ClpP-mGFPmut3 (f). ( g ) Live-cell HILO microscopy of cells expressing ClpP-mGFPmut3. Scale bars,1 μm.

    Techniques Used: Immunofluorescence, Microscopy, Fluorescence, Expressing, Labeling, Construct

    29) Product Images from "Genome-wide detection of conservative site-specific recombination in bacteria"

    Article Title: Genome-wide detection of conservative site-specific recombination in bacteria

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007332

    Expression of CDR20291_3128 is consistent with phase variation. Liquid culture samples from C . difficile R20291 and ∆3128::SNAP-tag were analyzed by light and fluorescence microscopy following staining with SNAP-Cell TMR-Star substrate. The left column shows the view of total cells in bright field light (BF), the middle column shows the SNAP-tag expressing cells pseudocolored in red, and the right column shows the merged pictures of BF and fluorescence.
    Figure Legend Snippet: Expression of CDR20291_3128 is consistent with phase variation. Liquid culture samples from C . difficile R20291 and ∆3128::SNAP-tag were analyzed by light and fluorescence microscopy following staining with SNAP-Cell TMR-Star substrate. The left column shows the view of total cells in bright field light (BF), the middle column shows the SNAP-tag expressing cells pseudocolored in red, and the right column shows the merged pictures of BF and fluorescence.

    Techniques Used: Expressing, Fluorescence, Microscopy, Staining

    30) Product Images from "The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin"

    Article Title: The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1219

    OTUD5 and UBR5 regulates the deposition of new Histone H2A at the damage sites. ( A ) Enlargement of SPT16 foci in UBR5 and OTUD5 knockdown cells. HeLa cells were irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters and fixed 1 hour after. The number of pixels in each γH2AX and SPT16 co-localized foci was measured using Image J, with each pixel represents an area of 0.2 μm 2 . The ratio of SPT16 to γH2AX was calculated by dividing the SPT16 area by the γH2AX area. The assay was performed in triplicates ( N = 35). Red bars indicate the median value for each set. ( B ) Ratio of SPT16 to Fok1 area in pTuner263 cells transfected with the siRNAs were quantified as in ( A ) The assay was performed in triplicates, N = 25. ( C ) Schematic for SNAP-H2A labeling and nucleosome integration. ( D ) U2OS cells stably expressing SNAP-H2A were first blocked with TMR Block and then irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters. Cells were labeled with TMR Star (red) and then fixed at indicated time points. Cells were counter-stained for γH2AX. The assay was performed in triplicates. Vector quantification of RFI of TMR signal (SNAP-H2A) ( N = 20); see ‘Materials and Methods’ section for details. Scale bars indicate 10 μm. (**** indicates P -value
    Figure Legend Snippet: OTUD5 and UBR5 regulates the deposition of new Histone H2A at the damage sites. ( A ) Enlargement of SPT16 foci in UBR5 and OTUD5 knockdown cells. HeLa cells were irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters and fixed 1 hour after. The number of pixels in each γH2AX and SPT16 co-localized foci was measured using Image J, with each pixel represents an area of 0.2 μm 2 . The ratio of SPT16 to γH2AX was calculated by dividing the SPT16 area by the γH2AX area. The assay was performed in triplicates ( N = 35). Red bars indicate the median value for each set. ( B ) Ratio of SPT16 to Fok1 area in pTuner263 cells transfected with the siRNAs were quantified as in ( A ) The assay was performed in triplicates, N = 25. ( C ) Schematic for SNAP-H2A labeling and nucleosome integration. ( D ) U2OS cells stably expressing SNAP-H2A were first blocked with TMR Block and then irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters. Cells were labeled with TMR Star (red) and then fixed at indicated time points. Cells were counter-stained for γH2AX. The assay was performed in triplicates. Vector quantification of RFI of TMR signal (SNAP-H2A) ( N = 20); see ‘Materials and Methods’ section for details. Scale bars indicate 10 μm. (**** indicates P -value

    Techniques Used: Irradiation, Transfection, Labeling, Stable Transfection, Expressing, Blocking Assay, Staining, Plasmid Preparation

    31) Product Images from "Lipids Regulate Lck Protein Activity through Their Interactions with the Lck Src Homology 2 Domain"

    Article Title: Lipids Regulate Lck Protein Activity through Their Interactions with the Lck Src Homology 2 Domain

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.720284

    Single molecule tracking of Lck and TCR-ζ in the TCR complex. A, representative images of Lck-GFP (WT and K182A/R184A) and SNAP-TMR-labeled TCR-ζ in a JCaM1.6 cell before and after OKT3 stimulation are shown. Green, red, and yellow lines
    Figure Legend Snippet: Single molecule tracking of Lck and TCR-ζ in the TCR complex. A, representative images of Lck-GFP (WT and K182A/R184A) and SNAP-TMR-labeled TCR-ζ in a JCaM1.6 cell before and after OKT3 stimulation are shown. Green, red, and yellow lines

    Techniques Used: Labeling

    32) Product Images from "CENP-A modifications on Ser68 and Lys124 are dispensable for establishment, maintenance, and long-term function of human centromeres"

    Article Title: CENP-A modifications on Ser68 and Lys124 are dispensable for establishment, maintenance, and long-term function of human centromeres

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2016.12.014

    Expression of CENP-A S68Q or CENP-A K124R (A) Schematic of the biallelic gene replacement approach used to replace the endogenous CENP-A gene with EGFP-AID-tagged CENP-A on one allele and CENP-A (wild type or mutant) tagged with SNAP-3xHA-P2A-NeoR on the other allele in DLD-1 TIR-1 cells via CRISPR/Cas9-mediated gene editing. (B) Schematic showing the indicated mutants of CENP-A tagged with SNAP-3xHA-P2A-NeoR, which replace the endogenous CENP-A gene on one allele, as indicated in Panel A. (C) Immunoblot of whole cell lysates from each of the indicated cell lines. Relevant cell lines were treated with 500 μM IAA for 24 hr to degrade EGFP-AID-tagged CENP-A. The blot was probed with anti-CENP-A and anti-tubulin antibodies. (D) Representative images showing localization of EGFP-AID-tagged CENP-A and CENP-A(wild type or mutant)-SNAP-3xHA at centromeres. Upon treatment with 500 μM IAA for 24 hr, the EGFP-AID-tagged CENP-A is no longer detected. (E) Quantification of the percentage of viable cells in the indicated cell lines upon treatment with 500 μM IAA for 8 d. Every 2 d, cells were collected and stained with Trypan Blue and counted on a hemocytometer to calculate the percentage of viable cells based on Trypan Blue uptake. Mean +/− SEM is shown for each time point. (F) Representative images of the indicated cell lines after 8 d of treatment with 500 μM IAA. The SNAP-3xHA-tagged CENP-A mutants are still present at endogenous centromeres. (G) Schematic for the quench-chase-pulse experiment in which the existing pool of CENP-A is quenched with SNAP-Cell Block, new CENP-A is synthesized, and newly loaded CENP-A is labeled with TMR- Star 24 hr later. (H) Quantification of the quench-chase-pulse experiment in which TMR- Star and total CENP-A signals are measured at centromeres in G1 cells (marked by a tubulin midbody remnant). Mean +/− SEM is shown. (I) Representative images showing that TMR- Star -labeled CENP-A is loaded at centromeres for each of the cell lines. The tubulin midbody remnant is shown between daughter G1 cells. Cells in which TMR- Star -labeled CENP-A is not detected at centromeres are shown in each representative image. Scale bar: 5 μm. Insets show magnification of the boxed region.
    Figure Legend Snippet: Expression of CENP-A S68Q or CENP-A K124R (A) Schematic of the biallelic gene replacement approach used to replace the endogenous CENP-A gene with EGFP-AID-tagged CENP-A on one allele and CENP-A (wild type or mutant) tagged with SNAP-3xHA-P2A-NeoR on the other allele in DLD-1 TIR-1 cells via CRISPR/Cas9-mediated gene editing. (B) Schematic showing the indicated mutants of CENP-A tagged with SNAP-3xHA-P2A-NeoR, which replace the endogenous CENP-A gene on one allele, as indicated in Panel A. (C) Immunoblot of whole cell lysates from each of the indicated cell lines. Relevant cell lines were treated with 500 μM IAA for 24 hr to degrade EGFP-AID-tagged CENP-A. The blot was probed with anti-CENP-A and anti-tubulin antibodies. (D) Representative images showing localization of EGFP-AID-tagged CENP-A and CENP-A(wild type or mutant)-SNAP-3xHA at centromeres. Upon treatment with 500 μM IAA for 24 hr, the EGFP-AID-tagged CENP-A is no longer detected. (E) Quantification of the percentage of viable cells in the indicated cell lines upon treatment with 500 μM IAA for 8 d. Every 2 d, cells were collected and stained with Trypan Blue and counted on a hemocytometer to calculate the percentage of viable cells based on Trypan Blue uptake. Mean +/− SEM is shown for each time point. (F) Representative images of the indicated cell lines after 8 d of treatment with 500 μM IAA. The SNAP-3xHA-tagged CENP-A mutants are still present at endogenous centromeres. (G) Schematic for the quench-chase-pulse experiment in which the existing pool of CENP-A is quenched with SNAP-Cell Block, new CENP-A is synthesized, and newly loaded CENP-A is labeled with TMR- Star 24 hr later. (H) Quantification of the quench-chase-pulse experiment in which TMR- Star and total CENP-A signals are measured at centromeres in G1 cells (marked by a tubulin midbody remnant). Mean +/− SEM is shown. (I) Representative images showing that TMR- Star -labeled CENP-A is loaded at centromeres for each of the cell lines. The tubulin midbody remnant is shown between daughter G1 cells. Cells in which TMR- Star -labeled CENP-A is not detected at centromeres are shown in each representative image. Scale bar: 5 μm. Insets show magnification of the boxed region.

    Techniques Used: Expressing, Mutagenesis, CRISPR, Staining, Blocking Assay, Synthesized, Labeling

    33) Product Images from "Distinct mechanistic responses to replication fork stalling induced by either nucleotide or protein deprivation"

    Article Title: Distinct mechanistic responses to replication fork stalling induced by either nucleotide or protein deprivation

    Journal: Cell Cycle

    doi: 10.1080/15384101.2017.1387696

    CHX does not immediately deplete replicative polymerases but disrupts new production of histones. (A) U2OS cells were either left untreated or treated with CHX for the timepoints indicated followed by Western blot probed with the specified antibodies. (B) Schematic protocol and representative images for fluorescent SNAP-tag labeling of U2OS cells stably expressing histone H3.1 or H3.3. Cells were quenched by DMSO (panel 1) or 0.2 μM blocking agent for 20 minutes (panel 2 and 3), labeled with 1 μM TMR for 30 minutes after a 3 hour release into fresh media +/- 10 μg/ml CHX. The contrast of the images in the middle panel of this figure has been equally enhanced in order to visualize the production of new histones after release from the quenching agent (panel 2), whereas the upper panel is unaltered. Furthermore, figure S4B and C (panel 3) shows cells without enhanced contrast, where arrows instead indicate new histone production. (C) Effect of HU on new histone H3.1 and H3.3 production via quenching of pre-existing histones using 0.2 μM blocking agent for 20 minutes, labeling with 1 μM TMR for 30 minutes after a 3 hour release into fresh media +/- 2 mM HU. (D) U2OS cells were treated with 10 μg/ml CHX or 2 mM HU for 24 hours and stained for RPA32. (E) U2OS cells were treated with 10 μg/ml CHX for 24 hours and co-stained for RPA32 and PML. See also Figure S5.
    Figure Legend Snippet: CHX does not immediately deplete replicative polymerases but disrupts new production of histones. (A) U2OS cells were either left untreated or treated with CHX for the timepoints indicated followed by Western blot probed with the specified antibodies. (B) Schematic protocol and representative images for fluorescent SNAP-tag labeling of U2OS cells stably expressing histone H3.1 or H3.3. Cells were quenched by DMSO (panel 1) or 0.2 μM blocking agent for 20 minutes (panel 2 and 3), labeled with 1 μM TMR for 30 minutes after a 3 hour release into fresh media +/- 10 μg/ml CHX. The contrast of the images in the middle panel of this figure has been equally enhanced in order to visualize the production of new histones after release from the quenching agent (panel 2), whereas the upper panel is unaltered. Furthermore, figure S4B and C (panel 3) shows cells without enhanced contrast, where arrows instead indicate new histone production. (C) Effect of HU on new histone H3.1 and H3.3 production via quenching of pre-existing histones using 0.2 μM blocking agent for 20 minutes, labeling with 1 μM TMR for 30 minutes after a 3 hour release into fresh media +/- 2 mM HU. (D) U2OS cells were treated with 10 μg/ml CHX or 2 mM HU for 24 hours and stained for RPA32. (E) U2OS cells were treated with 10 μg/ml CHX for 24 hours and co-stained for RPA32 and PML. See also Figure S5.

    Techniques Used: Western Blot, Labeling, Stable Transfection, Expressing, Blocking Assay, Staining

    34) Product Images from "Tousled-like kinases stabilize replication forks and show synthetic lethality with checkpoint and PARP inhibitors"

    Article Title: Tousled-like kinases stabilize replication forks and show synthetic lethality with checkpoint and PARP inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aat4985

    TLK2 is required for replication-coupled chromatin assembly. ( A ) Experimental design for assaying histone incorporation in cell lines stably expressing SNAP-tag histones H3.1 and H3.3. For quench (Q)–chase–pulse (P) experiments, U-2-OS cells were pulsed with SNAP-Block, chased for 7 hours, and then pulsed with TMR-Star. IF, immunofluorescence. ( B ) Quantification of SNAP-tag histone incorporation from n = 3 (H3.1) and n = 2 (H3.3) independent experiments as described in (A). Tetramethylrhodamine (TMR) intensity relative to mock-transfected cells is plotted. For each data point, n > 300 nuclei were analyzed. Means and SEM are indicated. For H3.1, a two-tailed t test was used for statistical analysis (* P
    Figure Legend Snippet: TLK2 is required for replication-coupled chromatin assembly. ( A ) Experimental design for assaying histone incorporation in cell lines stably expressing SNAP-tag histones H3.1 and H3.3. For quench (Q)–chase–pulse (P) experiments, U-2-OS cells were pulsed with SNAP-Block, chased for 7 hours, and then pulsed with TMR-Star. IF, immunofluorescence. ( B ) Quantification of SNAP-tag histone incorporation from n = 3 (H3.1) and n = 2 (H3.3) independent experiments as described in (A). Tetramethylrhodamine (TMR) intensity relative to mock-transfected cells is plotted. For each data point, n > 300 nuclei were analyzed. Means and SEM are indicated. For H3.1, a two-tailed t test was used for statistical analysis (* P

    Techniques Used: Stable Transfection, Expressing, Blocking Assay, Immunofluorescence, Transfection, Two Tailed Test

    35) Product Images from "Vimentin fibers orient traction stress"

    Article Title: Vimentin fibers orient traction stress

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1614610114

    ). ( A ) SNAP-TMR actin distribution in hFFs. Zoom-in of inset is the raw vimentin image overlaid with the cell outline. (Scale bars: 5 μm.) ( B ) Detected vimentin fibers overlaid with actin flow vectors (white). ( C ) Zoom-in of boxed regions in B . Vimentin fibers are shown in red. Each white pixel represents the position of an identified actin speckle. Vector length represents the time interval over which a speckle was tracked. ( D ) Actin flow speed vs. colocalized vimentin polymer organization (none, mesh, fiber) for n = 7 cells. Shown from left to right: no vimentin, M = 5,046 actin flow tracks; mesh, M = 14,130 flow tracks; and filamentous vimentin, M = 418 flow tracks.
    Figure Legend Snippet: ). ( A ) SNAP-TMR actin distribution in hFFs. Zoom-in of inset is the raw vimentin image overlaid with the cell outline. (Scale bars: 5 μm.) ( B ) Detected vimentin fibers overlaid with actin flow vectors (white). ( C ) Zoom-in of boxed regions in B . Vimentin fibers are shown in red. Each white pixel represents the position of an identified actin speckle. Vector length represents the time interval over which a speckle was tracked. ( D ) Actin flow speed vs. colocalized vimentin polymer organization (none, mesh, fiber) for n = 7 cells. Shown from left to right: no vimentin, M = 5,046 actin flow tracks; mesh, M = 14,130 flow tracks; and filamentous vimentin, M = 418 flow tracks.

    Techniques Used: Flow Cytometry, Plasmid Preparation

    36) Product Images from "Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings"

    Article Title: Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings

    Journal: bioRxiv

    doi: 10.1101/2020.03.09.983924

    SNAP-tag enabled in vivo imaging of tubulin in BY-2 cells and Arabidopsis a , Labeling mechanism of SNAP-tag. b , Cortical microtubules in SNAP-TUA5 expressing BY-2 cells with SNAP dyes denoted above images. Images show max intensity projection of confocal z-stack slices taken with 0.5 µm steps. c , Time-lapse imaging of mitotic microtubule dynamics TMR-star labeling of TUA5. Images taken every 30 sec, elapsed time (min) is shown. d , pUBQ10:SNAP-TUA5 and Col-0 (wildtype) seedlings were stained with 500 nM SNAP-Cell TMR-Star for 3h, lysed and analyzed by SDS-PAGE. Left panel: fluorescence; right panel Coomassie blue staining. e , Confocal images of mitotic cells in Arabidopsis root epidermis stained with TMR-Star. Spindles and phragmoplasts were observed. f , Root tip of Arabidopsis coexpressing p35S:YFP-LTI6b, p35:H2B-RFP, and pUBQ10:SNAP-TUA5. 3-day-old seedlings were incubated in 1/2MS containing 500 nM SNAP-Cell 647-SiR for 30 min. Scale bars: 10 µm. Experiments were repeated independently 3 times with comparable results.
    Figure Legend Snippet: SNAP-tag enabled in vivo imaging of tubulin in BY-2 cells and Arabidopsis a , Labeling mechanism of SNAP-tag. b , Cortical microtubules in SNAP-TUA5 expressing BY-2 cells with SNAP dyes denoted above images. Images show max intensity projection of confocal z-stack slices taken with 0.5 µm steps. c , Time-lapse imaging of mitotic microtubule dynamics TMR-star labeling of TUA5. Images taken every 30 sec, elapsed time (min) is shown. d , pUBQ10:SNAP-TUA5 and Col-0 (wildtype) seedlings were stained with 500 nM SNAP-Cell TMR-Star for 3h, lysed and analyzed by SDS-PAGE. Left panel: fluorescence; right panel Coomassie blue staining. e , Confocal images of mitotic cells in Arabidopsis root epidermis stained with TMR-Star. Spindles and phragmoplasts were observed. f , Root tip of Arabidopsis coexpressing p35S:YFP-LTI6b, p35:H2B-RFP, and pUBQ10:SNAP-TUA5. 3-day-old seedlings were incubated in 1/2MS containing 500 nM SNAP-Cell 647-SiR for 30 min. Scale bars: 10 µm. Experiments were repeated independently 3 times with comparable results.

    Techniques Used: In Vivo Imaging, Labeling, Expressing, Imaging, Staining, SDS Page, Fluorescence, Incubation

    37) Product Images from "Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG"

    Article Title: Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201205116

    Clathrin and pericentrin traffic along a shared route toward late-interphase centrosomes. (A) Asynchronous clone 3.3 cells were transiently transfected with GFP-pericentrin 48 h before labeling SNAP-uLCa with SNAP-TMR-Star. Cells were analyzed over 2 h by time-lapse confocal microscopy, taking 2D images every 10 min (37°C in 5% CO 2 at 95% humidity). Colocalization (yellow in merged images) between SNAP-TMR-Star–labeled SNAP-uLCa and GFP-pericentrin occurs at centrosomes (arrowheads) and adjacent submicrometer vesicles (arrows). Top right corners of merged images show time interval in minutes. Bar, 10 µm. Images are 3D maximum projections. (B) Asynchronous clone 3.3 cells transiently transfected with GFP-pericentrin and labeled with SNAP-TMR-Star were imaged by confocal microscopy. Colocalization of pericentrin and SNAP-uLCa is shown (yellow in merged image) at two separated centrosomes of a late-interphase cell and overlayed with the brightfield image. Bar, 10 µm. Images are 2D. (C) The cell shown in B was magnified twofold and analyzed by time-lapse confocal microscopy for 2 min with 10-s imaging intervals. SNAP-uLCa localizes with GFP-pericentrin at the centrosome, and pericentriolar SNAP-uLCa vesicles move toward the centrosome (arrowheads). Top right corners of merged images show the time interval. Bars, 5 µm. (insets) Boxed regions are magnified threefold. Bars, 2 µm. Images are 3D maximal projections.
    Figure Legend Snippet: Clathrin and pericentrin traffic along a shared route toward late-interphase centrosomes. (A) Asynchronous clone 3.3 cells were transiently transfected with GFP-pericentrin 48 h before labeling SNAP-uLCa with SNAP-TMR-Star. Cells were analyzed over 2 h by time-lapse confocal microscopy, taking 2D images every 10 min (37°C in 5% CO 2 at 95% humidity). Colocalization (yellow in merged images) between SNAP-TMR-Star–labeled SNAP-uLCa and GFP-pericentrin occurs at centrosomes (arrowheads) and adjacent submicrometer vesicles (arrows). Top right corners of merged images show time interval in minutes. Bar, 10 µm. Images are 3D maximum projections. (B) Asynchronous clone 3.3 cells transiently transfected with GFP-pericentrin and labeled with SNAP-TMR-Star were imaged by confocal microscopy. Colocalization of pericentrin and SNAP-uLCa is shown (yellow in merged image) at two separated centrosomes of a late-interphase cell and overlayed with the brightfield image. Bar, 10 µm. Images are 2D. (C) The cell shown in B was magnified twofold and analyzed by time-lapse confocal microscopy for 2 min with 10-s imaging intervals. SNAP-uLCa localizes with GFP-pericentrin at the centrosome, and pericentriolar SNAP-uLCa vesicles move toward the centrosome (arrowheads). Top right corners of merged images show the time interval. Bars, 5 µm. (insets) Boxed regions are magnified threefold. Bars, 2 µm. Images are 3D maximal projections.

    Techniques Used: Transfection, Labeling, Confocal Microscopy, Imaging

    38) Product Images from "Transcription-associated histone pruning demarcates macroH2A chromatin domains"

    Article Title: Transcription-associated histone pruning demarcates macroH2A chromatin domains

    Journal: Nature structural & molecular biology

    doi: 10.1038/s41594-018-0134-5

    Chromatin deposition of macroH2A variants is replication-independent. ( a ) Experimental scheme for in vivo labeling of newly incorporated SNAP-tagged histones. Histones expressed as SNAP-tag fusions in asynchronously growing cells were covalently bound by a non-fluorescent SNAP substrate, irreversibly blocking the existing histone pool from subsequent detection (quench). After allowing synthesis of new SNAP-tagged histones (chase), pulse-labeling with the fluorescent SNAP substrate TMR-Star exclusively marks newly synthesized histones (pulse). Pulse-only labeling represents the total level of old and new SNAP-tagged histones, and quench-pulse is used to control for the background fluorescence level. ( b ) FACS analysis of SNAP TMR signal normalized to DNA content (SNAPc) in nuclei isolated from asynchronous NIH 3T3 cells expressing SNAP-H3.1 or macroH2A2 (mH2A2)-SNAP. Nuclei positive for tagged histone incorporation (SNAPc+) were identified by comparison to quench-pulse cells. Insets show the percentage of SNAPc+ nuclei across the cell cycle. Cell cycle stages were identified by DNA content ( See Supplementary Fig. 1b ). ( c ) Microscopic analysis of newly synthesized H3.1-SNAP, macroH2A2-SNAP, macroH2A1.1 (mH2A1.1)-SNAP or macroH2A1.2 (mH2A1.2)-SNAP in the chromatin of asynchronous WT iDFs. EdU and Aurora B staining were used to distinguish cell cycle stages. Cells are scored as G1 (negative for EdU and Aurora B), S phase (EdU positive) or G2 (EdU negative, Aurora B positive). S phase is divided into early, mid and late phase based on the EdU distribution. Scale bar representing 10 μm applies to all panels. More than 50 cells were examined for each tagged histone.
    Figure Legend Snippet: Chromatin deposition of macroH2A variants is replication-independent. ( a ) Experimental scheme for in vivo labeling of newly incorporated SNAP-tagged histones. Histones expressed as SNAP-tag fusions in asynchronously growing cells were covalently bound by a non-fluorescent SNAP substrate, irreversibly blocking the existing histone pool from subsequent detection (quench). After allowing synthesis of new SNAP-tagged histones (chase), pulse-labeling with the fluorescent SNAP substrate TMR-Star exclusively marks newly synthesized histones (pulse). Pulse-only labeling represents the total level of old and new SNAP-tagged histones, and quench-pulse is used to control for the background fluorescence level. ( b ) FACS analysis of SNAP TMR signal normalized to DNA content (SNAPc) in nuclei isolated from asynchronous NIH 3T3 cells expressing SNAP-H3.1 or macroH2A2 (mH2A2)-SNAP. Nuclei positive for tagged histone incorporation (SNAPc+) were identified by comparison to quench-pulse cells. Insets show the percentage of SNAPc+ nuclei across the cell cycle. Cell cycle stages were identified by DNA content ( See Supplementary Fig. 1b ). ( c ) Microscopic analysis of newly synthesized H3.1-SNAP, macroH2A2-SNAP, macroH2A1.1 (mH2A1.1)-SNAP or macroH2A1.2 (mH2A1.2)-SNAP in the chromatin of asynchronous WT iDFs. EdU and Aurora B staining were used to distinguish cell cycle stages. Cells are scored as G1 (negative for EdU and Aurora B), S phase (EdU positive) or G2 (EdU negative, Aurora B positive). S phase is divided into early, mid and late phase based on the EdU distribution. Scale bar representing 10 μm applies to all panels. More than 50 cells were examined for each tagged histone.

    Techniques Used: In Vivo, Labeling, Blocking Assay, Synthesized, Fluorescence, FACS, Isolation, Expressing, Staining

    39) Product Images from "Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function"

    Article Title: Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.090639

    VP16, but not p65 tethering, negatively affects CENP-A loading at the HAC centromere. ( A ) Schematic diagram of quench–pulse-chase experiments in cells co-transfected with SNAP-tagged CENP-A and either of the tetR fusion constructs to determine the centromeric loading of newly synthesized CENP-A. ( B–D ) Cells expressing the indicated tetR fusion construct (green, panel 1) and pulse-labelled with TMR-Star (red, panel 2). Merged images (panel 3) represent the overlay of EYFP signal with TMR and DAPI staining (blue). Arrowheads depict the HAC. Scale bar: 5 μm. ( E ) Quantification of HAC-associated TMR-Star signal in cells as in B–D. AFU values are plotted relative to the average TMR-Star signal measured at endogenous centromeres. Solid lines indicate the median. ns, no significant difference.
    Figure Legend Snippet: VP16, but not p65 tethering, negatively affects CENP-A loading at the HAC centromere. ( A ) Schematic diagram of quench–pulse-chase experiments in cells co-transfected with SNAP-tagged CENP-A and either of the tetR fusion constructs to determine the centromeric loading of newly synthesized CENP-A. ( B–D ) Cells expressing the indicated tetR fusion construct (green, panel 1) and pulse-labelled with TMR-Star (red, panel 2). Merged images (panel 3) represent the overlay of EYFP signal with TMR and DAPI staining (blue). Arrowheads depict the HAC. Scale bar: 5 μm. ( E ) Quantification of HAC-associated TMR-Star signal in cells as in B–D. AFU values are plotted relative to the average TMR-Star signal measured at endogenous centromeres. Solid lines indicate the median. ns, no significant difference.

    Techniques Used: HAC Assay, Pulse Chase, Transfection, Construct, Synthesized, Expressing, Staining

    VP16 causes an increased rate of loss of CENP-A from the HAC centromere. ( A ) Schematic diagram of pulse-chase experiments in cells stably expressing SNAP-tagged CENP-A and transfected with the various tetR fusion constructs. ( B ) Quantification of HAC-associated TMR-Star signal in cells plotted relative to the average TMR-Star signal measured at ten endogenous centromeres. Solid lines indicate the median. ( C–E ) Cells expressing the indicated tetR fusion construct (green, panel 1) and pulse-labelled with TMR-Star (red, panel 2). Merged images (panel 3) represent the overlay of EYFP signal with TMR Star and DAPI staining (blue). Arrowheads depict the HAC. Scale bar: 5 μm.
    Figure Legend Snippet: VP16 causes an increased rate of loss of CENP-A from the HAC centromere. ( A ) Schematic diagram of pulse-chase experiments in cells stably expressing SNAP-tagged CENP-A and transfected with the various tetR fusion constructs. ( B ) Quantification of HAC-associated TMR-Star signal in cells plotted relative to the average TMR-Star signal measured at ten endogenous centromeres. Solid lines indicate the median. ( C–E ) Cells expressing the indicated tetR fusion construct (green, panel 1) and pulse-labelled with TMR-Star (red, panel 2). Merged images (panel 3) represent the overlay of EYFP signal with TMR Star and DAPI staining (blue). Arrowheads depict the HAC. Scale bar: 5 μm.

    Techniques Used: HAC Assay, Pulse Chase, Stable Transfection, Expressing, Transfection, Construct, Staining

    40) Product Images from "Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells"

    Article Title: Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078745

    Labelling of SNAP-, CLIP- and Halo-tagged proteins in chemically fixed and living budding yeast cells. (A) Chemically fixed cells expressing the respective self-labelling proteins targeted to the mitochondrial matrix (mtSNAP, mtCLIP, or mtHalo) were labelled. (B) Labelling of live yeast cells expressing the mitochondrial targeted self-labelling proteins using an electroporation protocol. (C) Live yeast cells expressing the indicated fusion proteins labelled by electroporation. Cells were labelled using commercially available TMR substrates. Yeast strains expressing Abp1-SNAP and Pil1-CLIP were created by epitope-tagging, while the other fusion constructs were plasmid encoded. Shown are maximum projections of confocal sections. Scale bar: 2 µm.
    Figure Legend Snippet: Labelling of SNAP-, CLIP- and Halo-tagged proteins in chemically fixed and living budding yeast cells. (A) Chemically fixed cells expressing the respective self-labelling proteins targeted to the mitochondrial matrix (mtSNAP, mtCLIP, or mtHalo) were labelled. (B) Labelling of live yeast cells expressing the mitochondrial targeted self-labelling proteins using an electroporation protocol. (C) Live yeast cells expressing the indicated fusion proteins labelled by electroporation. Cells were labelled using commercially available TMR substrates. Yeast strains expressing Abp1-SNAP and Pil1-CLIP were created by epitope-tagging, while the other fusion constructs were plasmid encoded. Shown are maximum projections of confocal sections. Scale bar: 2 µm.

    Techniques Used: Cross-linking Immunoprecipitation, Expressing, Electroporation, Construct, Plasmid Preparation

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    Article Snippet: .. Cell labeling or blocking was done by incubating cells with 2 µM SNAP-Cell TMR-Star (New England BioLabs) or 6.7 µM SNAP-Cell Block (New England Biolabs) diluted with media for 15 min, washed with phosphate-buffered saline (PBS) + 1 mM MgCl2 and CaCl2 , incubated in media for 1 h, washed with PBS + 1 mM MgCl2 and CaCl2 , and put back in media until ready to harvest. ..

    Article Title: CENP-A modifications on Ser68 and Lys124 are dispensable for establishment, maintenance, and long-term function of human centromeres
    Article Snippet: .. One coverslip was labeled with 2 μM SNAP-Cell TMR- Star (NEB, S9105S) for 30 min at 37°C, washed several times with warm growth medium, incubated in the growth medium supplemented with IAA for 2 hr, washed with warm growth medium again, and fixed in 4% formaldehyde to ensure that the block had gone to completion (t=0 time point). .. 24 hr after block, the remaining coverslips were labeled with 2 μM SNAP-Cell TMR- Star for 30 min at 37°C, washed several times with warm growth medium, incubated in the growth medium supplemented with IAA for 2 hr, washed with warm growth medium again, and fixed in 4% formaldehyde (t=24 hr time point).

    Concentration Assay:

    Article Title: The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin
    Article Snippet: .. Cells were labeled with SNAP-Cell TMR-Star (NEB) at 3 µM concentration immediately after UVC irradiation, to label the SNAP-H2A during the recovery time. .. Purified OTUD5 (5 μg) was mixed with Trypsin (Sigma Aldrich) at a 1:100 ratio of protease:protein and incubated in 100 mM Tris pH 8.5 for indicated times.

    Incubation:

    Article Title: High-resolution visualization of H3 variants during replication reveals their controlled recycling
    Article Snippet: .. For the pulse experiments, we incubated cells in complete medium containing 2 μM of SNAP-Cell TMR-Star (New England Biolabs) and 10 μM of EdU during 20 min for labeling. ..

    Article Title: Constitutive centromere-associated network contacts confer differential stability on CENP-A nucleosomes in vitro and in the cell
    Article Snippet: .. Cell labeling or blocking was done by incubating cells with 2 µM SNAP-Cell TMR-Star (New England BioLabs) or 6.7 µM SNAP-Cell Block (New England Biolabs) diluted with media for 15 min, washed with phosphate-buffered saline (PBS) + 1 mM MgCl2 and CaCl2 , incubated in media for 1 h, washed with PBS + 1 mM MgCl2 and CaCl2 , and put back in media until ready to harvest. ..

    Article Title: Visualizing the dynamic coupling of claudin strands to the actin cytoskeleton through ZO-1
    Article Snippet: .. Pulse-chase-pulse experiments Rat-1 cells stably expressing SNAP-tagged cldn2 cultured on glass coverslips were incubated with 3 μM SNAP-Cell TMR-star (all SNAP-cell reagents from New England Biolabs) for 30 min in medium and rinsed three times, and then SNAP-Cell block (10 μM) was added for 30 min. .. Cells were again washed three times and incubated for various periods of time (2–8 h) before addition of SNAP-Cell 647 SiR and incubation for 30 min.

    Article Title: POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication
    Article Snippet: .. For pulse-chase experiments, cells were incubated in medium containing 2 μM of SNAP-Cell TMR-Star for 20 min, washed twice with PBS, incubated in complete medium for 30 min and washed again twice with PBS. ..

    Article Title: Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3
    Article Snippet: .. To stain intracellular SNAP-tagged proteins with the standard protocol, benzylguanine (BG), conjugated to fluorophores (silicon rhodamine [SiR], or TMR-Star, commercially available as SNAP-Cell 647-SiR and SNAP-Cell TMR-Star [NEB]), was added to live cells and incubated for at least 15 min at 37°C, 5% CO2. .. This was followed by three washes in complete medium and an extended incubation in complete medium for at least 30 min to remove background fluorescence.

    Article Title: CENP-A modifications on Ser68 and Lys124 are dispensable for establishment, maintenance, and long-term function of human centromeres
    Article Snippet: .. One coverslip was labeled with 2 μM SNAP-Cell TMR- Star (NEB, S9105S) for 30 min at 37°C, washed several times with warm growth medium, incubated in the growth medium supplemented with IAA for 2 hr, washed with warm growth medium again, and fixed in 4% formaldehyde to ensure that the block had gone to completion (t=0 time point). .. 24 hr after block, the remaining coverslips were labeled with 2 μM SNAP-Cell TMR- Star for 30 min at 37°C, washed several times with warm growth medium, incubated in the growth medium supplemented with IAA for 2 hr, washed with warm growth medium again, and fixed in 4% formaldehyde (t=24 hr time point).

    Cell Culture:

    Article Title: Visualizing the dynamic coupling of claudin strands to the actin cytoskeleton through ZO-1
    Article Snippet: .. Pulse-chase-pulse experiments Rat-1 cells stably expressing SNAP-tagged cldn2 cultured on glass coverslips were incubated with 3 μM SNAP-Cell TMR-star (all SNAP-cell reagents from New England Biolabs) for 30 min in medium and rinsed three times, and then SNAP-Cell block (10 μM) was added for 30 min. .. Cells were again washed three times and incubated for various periods of time (2–8 h) before addition of SNAP-Cell 647 SiR and incubation for 30 min.

    Expressing:

    Article Title: Visualizing the dynamic coupling of claudin strands to the actin cytoskeleton through ZO-1
    Article Snippet: .. Pulse-chase-pulse experiments Rat-1 cells stably expressing SNAP-tagged cldn2 cultured on glass coverslips were incubated with 3 μM SNAP-Cell TMR-star (all SNAP-cell reagents from New England Biolabs) for 30 min in medium and rinsed three times, and then SNAP-Cell block (10 μM) was added for 30 min. .. Cells were again washed three times and incubated for various periods of time (2–8 h) before addition of SNAP-Cell 647 SiR and incubation for 30 min.

    Staining:

    Article Title: Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3
    Article Snippet: .. To stain intracellular SNAP-tagged proteins with the standard protocol, benzylguanine (BG), conjugated to fluorophores (silicon rhodamine [SiR], or TMR-Star, commercially available as SNAP-Cell 647-SiR and SNAP-Cell TMR-Star [NEB]), was added to live cells and incubated for at least 15 min at 37°C, 5% CO2. .. This was followed by three washes in complete medium and an extended incubation in complete medium for at least 30 min to remove background fluorescence.

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    New England Biolabs snap cell tmr star
    OTUD5 and UBR5 regulates the deposition of new Histone H2A at the damage sites. ( A ) Enlargement of SPT16 foci in UBR5 and OTUD5 knockdown cells. HeLa cells were irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters and fixed 1 hour after. The number of pixels in each γH2AX and SPT16 co-localized foci was measured using Image J, with each pixel represents an area of 0.2 μm 2 . The ratio of SPT16 to γH2AX was calculated by dividing the SPT16 area by the γH2AX area. The assay was performed in triplicates ( N = 35). Red bars indicate the median value for each set. ( B ) Ratio of SPT16 to Fok1 area in pTuner263 cells transfected with the siRNAs were quantified as in ( A ) The assay was performed in triplicates, N = 25. ( C ) Schematic for <t>SNAP-H2A</t> labeling and nucleosome integration. ( D ) U2OS cells stably expressing SNAP-H2A were first blocked with <t>TMR</t> Block and then irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters. Cells were labeled with TMR Star (red) and then fixed at indicated time points. Cells were counter-stained for γH2AX. The assay was performed in triplicates. Vector quantification of RFI of TMR signal (SNAP-H2A) ( N = 20); see ‘Materials and Methods’ section for details. Scale bars indicate 10 μm. (**** indicates P -value
    Snap Cell Tmr Star, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OTUD5 and UBR5 regulates the deposition of new Histone H2A at the damage sites. ( A ) Enlargement of SPT16 foci in UBR5 and OTUD5 knockdown cells. HeLa cells were irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters and fixed 1 hour after. The number of pixels in each γH2AX and SPT16 co-localized foci was measured using Image J, with each pixel represents an area of 0.2 μm 2 . The ratio of SPT16 to γH2AX was calculated by dividing the SPT16 area by the γH2AX area. The assay was performed in triplicates ( N = 35). Red bars indicate the median value for each set. ( B ) Ratio of SPT16 to Fok1 area in pTuner263 cells transfected with the siRNAs were quantified as in ( A ) The assay was performed in triplicates, N = 25. ( C ) Schematic for SNAP-H2A labeling and nucleosome integration. ( D ) U2OS cells stably expressing SNAP-H2A were first blocked with TMR Block and then irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters. Cells were labeled with TMR Star (red) and then fixed at indicated time points. Cells were counter-stained for γH2AX. The assay was performed in triplicates. Vector quantification of RFI of TMR signal (SNAP-H2A) ( N = 20); see ‘Materials and Methods’ section for details. Scale bars indicate 10 μm. (**** indicates P -value

    Journal: Nucleic Acids Research

    Article Title: The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin

    doi: 10.1093/nar/gky1219

    Figure Lengend Snippet: OTUD5 and UBR5 regulates the deposition of new Histone H2A at the damage sites. ( A ) Enlargement of SPT16 foci in UBR5 and OTUD5 knockdown cells. HeLa cells were irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters and fixed 1 hour after. The number of pixels in each γH2AX and SPT16 co-localized foci was measured using Image J, with each pixel represents an area of 0.2 μm 2 . The ratio of SPT16 to γH2AX was calculated by dividing the SPT16 area by the γH2AX area. The assay was performed in triplicates ( N = 35). Red bars indicate the median value for each set. ( B ) Ratio of SPT16 to Fok1 area in pTuner263 cells transfected with the siRNAs were quantified as in ( A ) The assay was performed in triplicates, N = 25. ( C ) Schematic for SNAP-H2A labeling and nucleosome integration. ( D ) U2OS cells stably expressing SNAP-H2A were first blocked with TMR Block and then irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters. Cells were labeled with TMR Star (red) and then fixed at indicated time points. Cells were counter-stained for γH2AX. The assay was performed in triplicates. Vector quantification of RFI of TMR signal (SNAP-H2A) ( N = 20); see ‘Materials and Methods’ section for details. Scale bars indicate 10 μm. (**** indicates P -value

    Article Snippet: Cells were labeled with SNAP-Cell TMR-Star (NEB) at 3 µM concentration immediately after UVC irradiation, to label the SNAP-H2A during the recovery time.

    Techniques: Irradiation, Transfection, Labeling, Stable Transfection, Expressing, Blocking Assay, Staining, Plasmid Preparation

    CENP-C and CENP-N degradation has no significant effect on centromeric CENP-A maintenance. (A, B) Cell lines containing AID tagged CENP-N, CENP-C, or both were treated with IAA to degrade the indicated proteins. Blue bars represent cells not treated with IAA. Cells were maintained in IAA beginning just after mitosis (red bars) and harvested in an early S-phase thymidine (thym) arrest (A) or after mitosis (mustard bars) or in early S phase (green bars) and harvested in a G2-phase roscovitine (rosco) arrest (B). Centromeric CENP-A immunofluorescence signal was normalized to the no-IAA signal. (C) Degradation of CENP-N inhibits new CENP-A assembly but not preexisting CENP-A in chromatin. The CENP-N AID-sfGFP cell line containing a stably integrated SNAP-tagged CENP-A was either fluorescently labeled or quenched according to the schematic (left panel). Green bars represent the time of synthesis of the fluorescent population of SNAP-tagged CENP-A. IAA was added as in A. Centromeric TMR-Star intensity represents the fluorescent population of SNAP-tagged CENP-A. Data are presented as mean ± SEM for three independent replicates. * p

    Journal: Molecular Biology of the Cell

    Article Title: Constitutive centromere-associated network contacts confer differential stability on CENP-A nucleosomes in vitro and in the cell

    doi: 10.1091/mbc.E17-10-0596

    Figure Lengend Snippet: CENP-C and CENP-N degradation has no significant effect on centromeric CENP-A maintenance. (A, B) Cell lines containing AID tagged CENP-N, CENP-C, or both were treated with IAA to degrade the indicated proteins. Blue bars represent cells not treated with IAA. Cells were maintained in IAA beginning just after mitosis (red bars) and harvested in an early S-phase thymidine (thym) arrest (A) or after mitosis (mustard bars) or in early S phase (green bars) and harvested in a G2-phase roscovitine (rosco) arrest (B). Centromeric CENP-A immunofluorescence signal was normalized to the no-IAA signal. (C) Degradation of CENP-N inhibits new CENP-A assembly but not preexisting CENP-A in chromatin. The CENP-N AID-sfGFP cell line containing a stably integrated SNAP-tagged CENP-A was either fluorescently labeled or quenched according to the schematic (left panel). Green bars represent the time of synthesis of the fluorescent population of SNAP-tagged CENP-A. IAA was added as in A. Centromeric TMR-Star intensity represents the fluorescent population of SNAP-tagged CENP-A. Data are presented as mean ± SEM for three independent replicates. * p

    Article Snippet: Cell labeling or blocking was done by incubating cells with 2 µM SNAP-Cell TMR-Star (New England BioLabs) or 6.7 µM SNAP-Cell Block (New England Biolabs) diluted with media for 15 min, washed with phosphate-buffered saline (PBS) + 1 mM MgCl2 and CaCl2 , incubated in media for 1 h, washed with PBS + 1 mM MgCl2 and CaCl2 , and put back in media until ready to harvest.

    Techniques: Immunofluorescence, Stable Transfection, Labeling

    (A) Cell lysates from stable CHIKV cells. Live cells were stained with BG-TMR-Star and lysed with Glasgow lysis buffer. The lysate was then bound to plastic chamber slides overnight and imaged the following day. Images were acquired with an LSM880 microscope operated in Fast Airyscan mode. Cell lysates from uninfected, naive HuH-7 cells are shown as a control. (B) Zoomed-in views of the sample shown in panel A, providing higher-magnification views of nsP3-containing granules and corresponding bright-field images (transmission). (C) Cell lysates from HuH-7 cells infected with CHIKV ZsGreen-P3 . Samples were prepared as described for panel A and imaged in the green channel. (D) Schematic overview of different populations (P1 to P4) obtained during culture of stable CHIKV cells. (E) Wide-field microscopy of stable CHIKV cells passaged for 1 week in the absence of puromycin (P2). Naive HuH-7 cells and cells treated for 1 week with puromycin (P1) are shown as controls. Images were obtained with an IncuCyte Zoom live-cell imaging system. (F) Effect of puromycin treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. Confluence of the two populations was determined from images taken with an IncuCyte Zoom live-cell imaging system. (G) Effect of sodium arsenite treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. To induce cellular stress granules, sodium arsenite was added for at least 30 min. Cells were fixed and then stained for SNAP-nsP3 (cyan) and G3BP2 (magenta). Stained cells were imaged by Airyscan microscopy. FOVs 1 and 3 were centered on cells expressing ZsGreen, whereas FOVs 2 and 4 focused on cells that were ZsGreen negative.

    Journal: Journal of Virology

    Article Title: Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3

    doi: 10.1128/JVI.00477-18

    Figure Lengend Snippet: (A) Cell lysates from stable CHIKV cells. Live cells were stained with BG-TMR-Star and lysed with Glasgow lysis buffer. The lysate was then bound to plastic chamber slides overnight and imaged the following day. Images were acquired with an LSM880 microscope operated in Fast Airyscan mode. Cell lysates from uninfected, naive HuH-7 cells are shown as a control. (B) Zoomed-in views of the sample shown in panel A, providing higher-magnification views of nsP3-containing granules and corresponding bright-field images (transmission). (C) Cell lysates from HuH-7 cells infected with CHIKV ZsGreen-P3 . Samples were prepared as described for panel A and imaged in the green channel. (D) Schematic overview of different populations (P1 to P4) obtained during culture of stable CHIKV cells. (E) Wide-field microscopy of stable CHIKV cells passaged for 1 week in the absence of puromycin (P2). Naive HuH-7 cells and cells treated for 1 week with puromycin (P1) are shown as controls. Images were obtained with an IncuCyte Zoom live-cell imaging system. (F) Effect of puromycin treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. Confluence of the two populations was determined from images taken with an IncuCyte Zoom live-cell imaging system. (G) Effect of sodium arsenite treatment on mixed populations containing both ZsGreen-positive and ZsGreen-negative cells. To induce cellular stress granules, sodium arsenite was added for at least 30 min. Cells were fixed and then stained for SNAP-nsP3 (cyan) and G3BP2 (magenta). Stained cells were imaged by Airyscan microscopy. FOVs 1 and 3 were centered on cells expressing ZsGreen, whereas FOVs 2 and 4 focused on cells that were ZsGreen negative.

    Article Snippet: To stain intracellular SNAP-tagged proteins with the standard protocol, benzylguanine (BG), conjugated to fluorophores (silicon rhodamine [SiR], or TMR-Star, commercially available as SNAP-Cell 647-SiR and SNAP-Cell TMR-Star [NEB]), was added to live cells and incubated for at least 15 min at 37°C, 5% CO2.

    Techniques: Staining, Lysis, Microscopy, Transmission Assay, Infection, Live Cell Imaging, Expressing

    Hat1 knock-down causes decreased CENP-A loading. ( A ) Experimental design of pulse labeling-mediated analysis of CENP-A centromeric loading. ( B ) RNAi efficiency was determined by RT-qPCR for Hat1. Transcript levels for Hat1, CENP-A and histone H4 in Hat1 RNAi cells were normalized against GAPDH and are expressed relative to those of the respective genes in TetR-treated S2 cells (control). ( C ) Example image of reduced CENP-A intensities in Hat1 RNAi cells. Cells were processed according to the scheme in (A) and newly loaded SNAP-CENP-A was visualized by staining with TMR-Star. Images were acquired and processed with identical settings. ( D ) Quantification of SNAP-CENP-A intensities in Hat1 knock-down and TetR RNAi control cells using Imaris v5.1 software. Statistical significance was determined by unpaired t -test and Mann–Whitney test (*** P

    Journal: Nucleic Acids Research

    Article Title: A novel role for the histone acetyltransferase Hat1 in the CENP-A/CID assembly pathway in Drosophila melanogaster

    doi: 10.1093/nar/gkv1235

    Figure Lengend Snippet: Hat1 knock-down causes decreased CENP-A loading. ( A ) Experimental design of pulse labeling-mediated analysis of CENP-A centromeric loading. ( B ) RNAi efficiency was determined by RT-qPCR for Hat1. Transcript levels for Hat1, CENP-A and histone H4 in Hat1 RNAi cells were normalized against GAPDH and are expressed relative to those of the respective genes in TetR-treated S2 cells (control). ( C ) Example image of reduced CENP-A intensities in Hat1 RNAi cells. Cells were processed according to the scheme in (A) and newly loaded SNAP-CENP-A was visualized by staining with TMR-Star. Images were acquired and processed with identical settings. ( D ) Quantification of SNAP-CENP-A intensities in Hat1 knock-down and TetR RNAi control cells using Imaris v5.1 software. Statistical significance was determined by unpaired t -test and Mann–Whitney test (*** P

    Article Snippet: After 48 h (Figure , Supplementary Figure S6) or 24 h (Supplementary Figure S6) chase, cells were pulse-labeled with 4.5 μM SNAP-Cell TMR Star (NEB) for 30 min. Nonreacted TMR Star was washed out, cells were fixed in 3.7% paraformaldehyde/0.3% Triton-X100 and nuclei were counterstained with DAPI.

    Techniques: Labeling, Quantitative RT-PCR, Staining, Software, MANN-WHITNEY