u2os cells  (Jena Bioscience)


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    Name:
    5 Ethynyl uridine 5 EU
    Description:
    Ethynyl labeled uridine 5 EU can be used as a replacement for BrU 5 Bromo uridine to measure de novo RNA synthesis in proliferating cells 5 EU is cell permeable and incorporates into nascent RNA instead of its natural analog uridine The resulting ethynyl functionalized RNA can subsequently be detected via Cu I catalyzed click chemistry that offers the choice to introduce a Biotin group via Azides of Biotin for subsequent purification tasks or a fluorescent group via Azides of fluorescent dyes for subsequent microscopic imaging 1 Presolski et al 2 and Hong et al 3 provide a general protocol for Cu I catalyzed click chemistry reactions that may be used as a starting point for the set up and optimization of individual assays
    Catalog Number:
    clk-n002-10
    Molecular Weight:
    268.22 g/mol
    Price:
    102.9
    Applications:
    RNA synthesis monitoring[1]
    Purity:
    ≥ 99 % (HPLC)
    Category:
    Click Chemistry
    Format:
    white to off-white solid
    Formula:
    C11H12N2O6
    Buy from Supplier


    Structured Review

    Jena Bioscience u2os cells
    Cancer cells with chronic BRG1 deficiency restore GTF2H1 expression. a Immunoblot showing total protein levels of BRM, BRG1, and GTF2H1, in cell lysates of <t>U2OS</t> and BRG1-deficient non-small lung cancer cell (NSCLC) lines A549 and H1299 treated with control (CTRL), BRG1 or BRM siRNAs. Ku70 was used as loading control. b Relative quantification of GTF2H1 protein levels in U2OS, A549, and H1299 cells transfected with control (CTRL), BRG1 or BRM siRNA. GTF2H1 levels were normalized to Ku70 and the total relative amount of GTF2H1 in whole cell lysates was set to 1.0 in U2OS siCTRL. Mean S.E.M. from at least three independent experiments ** P
    Ethynyl labeled uridine 5 EU can be used as a replacement for BrU 5 Bromo uridine to measure de novo RNA synthesis in proliferating cells 5 EU is cell permeable and incorporates into nascent RNA instead of its natural analog uridine The resulting ethynyl functionalized RNA can subsequently be detected via Cu I catalyzed click chemistry that offers the choice to introduce a Biotin group via Azides of Biotin for subsequent purification tasks or a fluorescent group via Azides of fluorescent dyes for subsequent microscopic imaging 1 Presolski et al 2 and Hong et al 3 provide a general protocol for Cu I catalyzed click chemistry reactions that may be used as a starting point for the set up and optimization of individual assays
    https://www.bioz.com/result/u2os cells/product/Jena Bioscience
    Average 92 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    u2os cells - by Bioz Stars, 2020-09
    92/100 stars

    Images

    1) Product Images from "DNA damage sensitivity of SWI/SNF-deficient cells depends on TFIIH subunit p62/GTF2H1"

    Article Title: DNA damage sensitivity of SWI/SNF-deficient cells depends on TFIIH subunit p62/GTF2H1

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06402-y

    Cancer cells with chronic BRG1 deficiency restore GTF2H1 expression. a Immunoblot showing total protein levels of BRM, BRG1, and GTF2H1, in cell lysates of U2OS and BRG1-deficient non-small lung cancer cell (NSCLC) lines A549 and H1299 treated with control (CTRL), BRG1 or BRM siRNAs. Ku70 was used as loading control. b Relative quantification of GTF2H1 protein levels in U2OS, A549, and H1299 cells transfected with control (CTRL), BRG1 or BRM siRNA. GTF2H1 levels were normalized to Ku70 and the total relative amount of GTF2H1 in whole cell lysates was set to 1.0 in U2OS siCTRL. Mean S.E.M. from at least three independent experiments ** P
    Figure Legend Snippet: Cancer cells with chronic BRG1 deficiency restore GTF2H1 expression. a Immunoblot showing total protein levels of BRM, BRG1, and GTF2H1, in cell lysates of U2OS and BRG1-deficient non-small lung cancer cell (NSCLC) lines A549 and H1299 treated with control (CTRL), BRG1 or BRM siRNAs. Ku70 was used as loading control. b Relative quantification of GTF2H1 protein levels in U2OS, A549, and H1299 cells transfected with control (CTRL), BRG1 or BRM siRNA. GTF2H1 levels were normalized to Ku70 and the total relative amount of GTF2H1 in whole cell lysates was set to 1.0 in U2OS siCTRL. Mean S.E.M. from at least three independent experiments ** P

    Techniques Used: Expressing, Transfection

    GTF2H1 expression rescues TFIIH in BRM/BRG1 depleted cells. Representative IF of XPD recruitment (red channel) to LUD marked by XPC (cyan channel). U2OS cells were fixed 30 min after local UV-C irradiation (60 J/m 2 ) through a microporous membrane (8 µm). a U2OS cells were treated with control (CTRL), BRM, or GTF2H1 siRNAs and transiently transfected with TFIIH subunits XPB or GTF2H1 fused to GFP (green channel). Scale bar: 10 µm. b Quantification of XPD recruitment to LUD. Relative accumulation at LUD (over nuclear background) in each condition was normalized to control (siCTRL without transient transfection of TFIIH subunits, indicated by “empty“ symbol), in which nuclear background was set at 0 and maximal signal at LUD set to 1.0 ( > 50 cells per sample, mean S.E.M. from four independent experiments). *** P
    Figure Legend Snippet: GTF2H1 expression rescues TFIIH in BRM/BRG1 depleted cells. Representative IF of XPD recruitment (red channel) to LUD marked by XPC (cyan channel). U2OS cells were fixed 30 min after local UV-C irradiation (60 J/m 2 ) through a microporous membrane (8 µm). a U2OS cells were treated with control (CTRL), BRM, or GTF2H1 siRNAs and transiently transfected with TFIIH subunits XPB or GTF2H1 fused to GFP (green channel). Scale bar: 10 µm. b Quantification of XPD recruitment to LUD. Relative accumulation at LUD (over nuclear background) in each condition was normalized to control (siCTRL without transient transfection of TFIIH subunits, indicated by “empty“ symbol), in which nuclear background was set at 0 and maximal signal at LUD set to 1.0 ( > 50 cells per sample, mean S.E.M. from four independent experiments). *** P

    Techniques Used: Expressing, Irradiation, Transfection

    BRM stabilizes TFIIH by promoting GTF2H1 expression. a Relative quantification of individual TFIIH genes expression in U2OS cells treated with control (CTRL) or BRM siRNAs, as determined with RT-qPCR. Individual basal gene expression in BRM knockdown was normalized to siCTRL levels, which were set to 1.0 (dotted line in graph). GAPDH expression was used for normalization. Mean S.E.M. of at least three independent experiments. ** P
    Figure Legend Snippet: BRM stabilizes TFIIH by promoting GTF2H1 expression. a Relative quantification of individual TFIIH genes expression in U2OS cells treated with control (CTRL) or BRM siRNAs, as determined with RT-qPCR. Individual basal gene expression in BRM knockdown was normalized to siCTRL levels, which were set to 1.0 (dotted line in graph). GAPDH expression was used for normalization. Mean S.E.M. of at least three independent experiments. ** P

    Techniques Used: Expressing, Quantitative RT-PCR

    2) Product Images from "γH2AX in the S Phase After UV Irradiation Corresponds to the Sites of DNA Replication and Not DNA Damage"

    Article Title: γH2AX in the S Phase After UV Irradiation Corresponds to the Sites of DNA Replication and Not DNA Damage

    Journal: bioRxiv

    doi: 10.1101/810689

    UV-induced γH2AX does not report on the extent of DNA damage in the S phase cells (A) Definition of the colocalization metric: more than 50% overlap between the foci from the two channels at a position. Images are of NCS-treated cells: those 53BP1 foci colocalizing with γH2AX foci are considered as DSBs (B) Percentage of γH2AX foci which are double strand breaks for control, UV- and NCS-treated cells. For UV treatment a very little fraction of γH2AX foci in the S phase cells corresponds to DSBs. (C) Global transcription is measured in UV-treated cells by quantifying 5-ethynyl-uridine incorporation. (D) Bar graphs for γH2AX and EU are normalized across the four dosages. Resultant bar graphs for EU are inverted over those for γH2AX such that the total height of the two bars corresponding to control population in every cell cycle phase is unity. A clear gap is observed between γH2AX and EU bars for G1 and G2/M phases after UV treatment. The γH2AX bars in the S phase not just overlap with EU bars but goes past the unit box showing a disproportionate increase in γH2AX. (E) Mean levels of γH2AX increase with the increase in DNA damage as achieved by increasing the UV dosage in all the phases of the cell cycle. For all the dosages, there is a sharp γH2AX peak in the S phase. (F) Increase in DNA damage always leads to decrease in EU as seen across the population treated with different dosages of UV. But within a population there is no dip in EU levels corresponding to the γH2AX peak showing that γH2AX in the S phase after UV does not reflect on the total extent of DNA damage in those cells. Cells were treated with 10 J/m 2 and 1.6 µg/ml NCS for two minutes. Line graphs are showing relative levels with respect to the mean value of G1 phase in control cells. Scale bar: 10 µm.
    Figure Legend Snippet: UV-induced γH2AX does not report on the extent of DNA damage in the S phase cells (A) Definition of the colocalization metric: more than 50% overlap between the foci from the two channels at a position. Images are of NCS-treated cells: those 53BP1 foci colocalizing with γH2AX foci are considered as DSBs (B) Percentage of γH2AX foci which are double strand breaks for control, UV- and NCS-treated cells. For UV treatment a very little fraction of γH2AX foci in the S phase cells corresponds to DSBs. (C) Global transcription is measured in UV-treated cells by quantifying 5-ethynyl-uridine incorporation. (D) Bar graphs for γH2AX and EU are normalized across the four dosages. Resultant bar graphs for EU are inverted over those for γH2AX such that the total height of the two bars corresponding to control population in every cell cycle phase is unity. A clear gap is observed between γH2AX and EU bars for G1 and G2/M phases after UV treatment. The γH2AX bars in the S phase not just overlap with EU bars but goes past the unit box showing a disproportionate increase in γH2AX. (E) Mean levels of γH2AX increase with the increase in DNA damage as achieved by increasing the UV dosage in all the phases of the cell cycle. For all the dosages, there is a sharp γH2AX peak in the S phase. (F) Increase in DNA damage always leads to decrease in EU as seen across the population treated with different dosages of UV. But within a population there is no dip in EU levels corresponding to the γH2AX peak showing that γH2AX in the S phase after UV does not reflect on the total extent of DNA damage in those cells. Cells were treated with 10 J/m 2 and 1.6 µg/ml NCS for two minutes. Line graphs are showing relative levels with respect to the mean value of G1 phase in control cells. Scale bar: 10 µm.

    Techniques Used:

    3) Product Images from "Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei"

    Article Title: Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1255

    RHS are involved in global mRNA synthesis ( A ) Quantification of 5-ethynyl uridine (5-EU) incorporation. Each RNAi line was cultured in the presence or absence of tetracycline for 2 days and pulsed for 10 min with 5-EU ( n = 3). Values are given as a percentage of the uninduced control. P-values are shown for paired, one-tailed t-tests. Error bars = SD. ( B ) Correlation between reads per million (RPM) for nascent mRNAs from RNAi lines cultured in the presence or absence of tetracycline for 2 days. R: Pearson correlation coefficient. Biological replicates were performed. See also Supplemental Figure S5 .
    Figure Legend Snippet: RHS are involved in global mRNA synthesis ( A ) Quantification of 5-ethynyl uridine (5-EU) incorporation. Each RNAi line was cultured in the presence or absence of tetracycline for 2 days and pulsed for 10 min with 5-EU ( n = 3). Values are given as a percentage of the uninduced control. P-values are shown for paired, one-tailed t-tests. Error bars = SD. ( B ) Correlation between reads per million (RPM) for nascent mRNAs from RNAi lines cultured in the presence or absence of tetracycline for 2 days. R: Pearson correlation coefficient. Biological replicates were performed. See also Supplemental Figure S5 .

    Techniques Used: Cell Culture, One-tailed Test

    4) Product Images from "γH2AX in the S Phase After UV Irradiation Corresponds to the Sites of DNA Replication and Not DNA Damage"

    Article Title: γH2AX in the S Phase After UV Irradiation Corresponds to the Sites of DNA Replication and Not DNA Damage

    Journal: bioRxiv

    doi: 10.1101/810689

    UV-induced γH2AX does not report on the extent of DNA damage in the S phase cells (A) Definition of the colocalization metric: more than 50% overlap between the foci from the two channels at a position. Images are of NCS-treated cells: those 53BP1 foci colocalizing with γH2AX foci are considered as DSBs (B) Percentage of γH2AX foci which are double strand breaks for control, UV- and NCS-treated cells. For UV treatment a very little fraction of γH2AX foci in the S phase cells corresponds to DSBs. (C) Global transcription is measured in UV-treated cells by quantifying 5-ethynyl-uridine incorporation. (D) Bar graphs for γH2AX and EU are normalized across the four dosages. Resultant bar graphs for EU are inverted over those for γH2AX such that the total height of the two bars corresponding to control population in every cell cycle phase is unity. A clear gap is observed between γH2AX and EU bars for G1 and G2/M phases after UV treatment. The γH2AX bars in the S phase not just overlap with EU bars but goes past the unit box showing a disproportionate increase in γH2AX. (E) Mean levels of γH2AX increase with the increase in DNA damage as achieved by increasing the UV dosage in all the phases of the cell cycle. For all the dosages, there is a sharp γH2AX peak in the S phase. (F) Increase in DNA damage always leads to decrease in EU as seen across the population treated with different dosages of UV. But within a population there is no dip in EU levels corresponding to the γH2AX peak showing that γH2AX in the S phase after UV does not reflect on the total extent of DNA damage in those cells. Cells were treated with 10 J/m 2 and 1.6 µg/ml NCS for two minutes. Line graphs are showing relative levels with respect to the mean value of G1 phase in control cells. Scale bar: 10 µm.
    Figure Legend Snippet: UV-induced γH2AX does not report on the extent of DNA damage in the S phase cells (A) Definition of the colocalization metric: more than 50% overlap between the foci from the two channels at a position. Images are of NCS-treated cells: those 53BP1 foci colocalizing with γH2AX foci are considered as DSBs (B) Percentage of γH2AX foci which are double strand breaks for control, UV- and NCS-treated cells. For UV treatment a very little fraction of γH2AX foci in the S phase cells corresponds to DSBs. (C) Global transcription is measured in UV-treated cells by quantifying 5-ethynyl-uridine incorporation. (D) Bar graphs for γH2AX and EU are normalized across the four dosages. Resultant bar graphs for EU are inverted over those for γH2AX such that the total height of the two bars corresponding to control population in every cell cycle phase is unity. A clear gap is observed between γH2AX and EU bars for G1 and G2/M phases after UV treatment. The γH2AX bars in the S phase not just overlap with EU bars but goes past the unit box showing a disproportionate increase in γH2AX. (E) Mean levels of γH2AX increase with the increase in DNA damage as achieved by increasing the UV dosage in all the phases of the cell cycle. For all the dosages, there is a sharp γH2AX peak in the S phase. (F) Increase in DNA damage always leads to decrease in EU as seen across the population treated with different dosages of UV. But within a population there is no dip in EU levels corresponding to the γH2AX peak showing that γH2AX in the S phase after UV does not reflect on the total extent of DNA damage in those cells. Cells were treated with 10 J/m 2 and 1.6 µg/ml NCS for two minutes. Line graphs are showing relative levels with respect to the mean value of G1 phase in control cells. Scale bar: 10 µm.

    Techniques Used:

    5) Product Images from "Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei"

    Article Title: Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1255

    RHS are involved in global mRNA synthesis ( A ) Quantification of 5-ethynyl uridine (5-EU) incorporation. Each RNAi line was cultured in the presence or absence of tetracycline for 2 days and pulsed for 10 min with 5-EU ( n = 3). Values are given as a percentage of the uninduced control. P-values are shown for paired, one-tailed t-tests. Error bars = SD. ( B ) Correlation between reads per million (RPM) for nascent mRNAs from RNAi lines cultured in the presence or absence of tetracycline for 2 days. R: Pearson correlation coefficient. Biological replicates were performed. See also Supplemental Figure S5 .
    Figure Legend Snippet: RHS are involved in global mRNA synthesis ( A ) Quantification of 5-ethynyl uridine (5-EU) incorporation. Each RNAi line was cultured in the presence or absence of tetracycline for 2 days and pulsed for 10 min with 5-EU ( n = 3). Values are given as a percentage of the uninduced control. P-values are shown for paired, one-tailed t-tests. Error bars = SD. ( B ) Correlation between reads per million (RPM) for nascent mRNAs from RNAi lines cultured in the presence or absence of tetracycline for 2 days. R: Pearson correlation coefficient. Biological replicates were performed. See also Supplemental Figure S5 .

    Techniques Used: Cell Culture, One-tailed Test

    Related Articles

    Isolation:

    Article Title: Hormone stimulation of androgen receptor mediates dynamic changes in DNA methylation patterns at regulatory elements
    Article Snippet: .. The cells were pulsed with 0.5 mM 5-ethynyl uridine (5-EU) (Jena Biosciences, Cat #CLK-N002-10) for 30 min before harvesting in 1.0 ml Trizol® reagent for RNA isolation. ..

    other:

    Article Title: Visualization of the Nucleolus Using Ethynyl Uridine
    Article Snippet: In such cases, the use of CLK-N002-10 product (diluted in water) is recommended.

    Labeling:

    Article Title: Visualization of the Nucleolus Using Ethynyl Uridine
    Article Snippet: .. EU Labeling Two types of EU were used in this study, product CLK-N002-10 (Jena Bioscience, 200 mM in sterile water) and E-10345 (Life Technologies, 100 mM in DMSO). .. Four days old A. thaliana seedlings were transferred into 12-well plates (Greiner Bio-One).

    Cell Culture:

    Article Title: The Dynamics of Cytoplasmic mRNA Metabolism.
    Article Snippet: .. Metabolic-Labeling Time Courses Cells from each line were plated onto 500 cm2 plates at 6.6 million cells per plate and cultured for two days such that they reached 70%–80% confluency, at which point growth media was supplemented with 5-ethynyl uridine (5EU, Jena Biosciences) (Jao and Salic, 2008) at a final concentration of 400 mM. .. After the desired labeling intervals cells were harvested (Figure 1A).

    Concentration Assay:

    Article Title: The Dynamics of Cytoplasmic mRNA Metabolism.
    Article Snippet: .. Metabolic-Labeling Time Courses Cells from each line were plated onto 500 cm2 plates at 6.6 million cells per plate and cultured for two days such that they reached 70%–80% confluency, at which point growth media was supplemented with 5-ethynyl uridine (5EU, Jena Biosciences) (Jao and Salic, 2008) at a final concentration of 400 mM. .. After the desired labeling intervals cells were harvested (Figure 1A).

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  • 88
    Jena Bioscience 5 ethynyl uridine 5 eu
    UV-induced γH2AX does not report on the extent of DNA damage in the S phase cells (A) Definition of the colocalization metric: more than 50% overlap between the foci from the two channels at a position. Images are of NCS-treated cells: those 53BP1 foci colocalizing with γH2AX foci are considered as DSBs (B) Percentage of γH2AX foci which are double strand breaks for control, UV- and NCS-treated cells. For UV treatment a very little fraction of γH2AX foci in the S phase cells corresponds to DSBs. (C) Global transcription is measured in UV-treated cells by quantifying <t>5-ethynyl-uridine</t> incorporation. (D) Bar graphs for γH2AX and EU are normalized across the four dosages. Resultant bar graphs for EU are inverted over those for γH2AX such that the total height of the two bars corresponding to control population in every cell cycle phase is unity. A clear gap is observed between γH2AX and EU bars for G1 and G2/M phases after UV treatment. The γH2AX bars in the S phase not just overlap with EU bars but goes past the unit box showing a disproportionate increase in γH2AX. (E) Mean levels of γH2AX increase with the increase in DNA damage as achieved by increasing the UV dosage in all the phases of the cell cycle. For all the dosages, there is a sharp γH2AX peak in the S phase. (F) Increase in DNA damage always leads to decrease in EU as seen across the population treated with different dosages of UV. But within a population there is no dip in EU levels corresponding to the γH2AX peak showing that γH2AX in the S phase after UV does not reflect on the total extent of DNA damage in those cells. Cells were treated with 10 J/m 2 and 1.6 µg/ml NCS for two minutes. Line graphs are showing relative levels with respect to the mean value of G1 phase in control cells. Scale bar: 10 µm.
    5 Ethynyl Uridine 5 Eu, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/5 ethynyl uridine 5 eu/product/Jena Bioscience
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    5 ethynyl uridine 5 eu - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    84
    Jena Bioscience 5 ethyniluridine eu incorporation
    1,6-HD compromises LLPS in living human cells. ( A ) HeLa cells were untreated or treated with Tween 20 (1%, 10 min; “Tween”), Tween 20 followed with 1,6-HD (5%, 15 min; “1,6-HD”) before being analyzed using CellEvent Caspase 3/7 Detection Reagent. The cells treated as described and then incubated in fresh culture medium for 1.5 h were analysed as well. Percent of caspase 3/7-positive (apoptotic) cells is shown. ( B ) HeLa cells treated as described in ( A ) were pulsed with <t>5-ethyniluridine</t> (EU, 200 μM, 15 min). Box plots show the EU fluorescence intensities. Horizontal lines represent the medians. ( C ) HeLa cells transfected with pHR-FUSN-mCh-Cry2WT were transiently permeabilized and then either mock-treated (control), treated with 1,6-HD (5%, 15 min), or treated with 1,6-HD and allowed to recover for 1.5 h. OptoDroplet formation was monitored as described in ( Shin et al. 2017 ). ( D ) Transiently permeabilized HeLa cells were untreated (control), treated with 1,6-HD (5%, 15 min), or treated with 1,6-HD and allowed to recover for 1.5 h before being stained for coilin (red).
    5 Ethyniluridine Eu Incorporation, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/5 ethyniluridine eu incorporation/product/Jena Bioscience
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    5 ethyniluridine eu incorporation - by Bioz Stars, 2020-09
    84/100 stars
      Buy from Supplier

    Image Search Results


    UV-induced γH2AX does not report on the extent of DNA damage in the S phase cells (A) Definition of the colocalization metric: more than 50% overlap between the foci from the two channels at a position. Images are of NCS-treated cells: those 53BP1 foci colocalizing with γH2AX foci are considered as DSBs (B) Percentage of γH2AX foci which are double strand breaks for control, UV- and NCS-treated cells. For UV treatment a very little fraction of γH2AX foci in the S phase cells corresponds to DSBs. (C) Global transcription is measured in UV-treated cells by quantifying 5-ethynyl-uridine incorporation. (D) Bar graphs for γH2AX and EU are normalized across the four dosages. Resultant bar graphs for EU are inverted over those for γH2AX such that the total height of the two bars corresponding to control population in every cell cycle phase is unity. A clear gap is observed between γH2AX and EU bars for G1 and G2/M phases after UV treatment. The γH2AX bars in the S phase not just overlap with EU bars but goes past the unit box showing a disproportionate increase in γH2AX. (E) Mean levels of γH2AX increase with the increase in DNA damage as achieved by increasing the UV dosage in all the phases of the cell cycle. For all the dosages, there is a sharp γH2AX peak in the S phase. (F) Increase in DNA damage always leads to decrease in EU as seen across the population treated with different dosages of UV. But within a population there is no dip in EU levels corresponding to the γH2AX peak showing that γH2AX in the S phase after UV does not reflect on the total extent of DNA damage in those cells. Cells were treated with 10 J/m 2 and 1.6 µg/ml NCS for two minutes. Line graphs are showing relative levels with respect to the mean value of G1 phase in control cells. Scale bar: 10 µm.

    Journal: bioRxiv

    Article Title: γH2AX in the S Phase After UV Irradiation Corresponds to the Sites of DNA Replication and Not DNA Damage

    doi: 10.1101/810689

    Figure Lengend Snippet: UV-induced γH2AX does not report on the extent of DNA damage in the S phase cells (A) Definition of the colocalization metric: more than 50% overlap between the foci from the two channels at a position. Images are of NCS-treated cells: those 53BP1 foci colocalizing with γH2AX foci are considered as DSBs (B) Percentage of γH2AX foci which are double strand breaks for control, UV- and NCS-treated cells. For UV treatment a very little fraction of γH2AX foci in the S phase cells corresponds to DSBs. (C) Global transcription is measured in UV-treated cells by quantifying 5-ethynyl-uridine incorporation. (D) Bar graphs for γH2AX and EU are normalized across the four dosages. Resultant bar graphs for EU are inverted over those for γH2AX such that the total height of the two bars corresponding to control population in every cell cycle phase is unity. A clear gap is observed between γH2AX and EU bars for G1 and G2/M phases after UV treatment. The γH2AX bars in the S phase not just overlap with EU bars but goes past the unit box showing a disproportionate increase in γH2AX. (E) Mean levels of γH2AX increase with the increase in DNA damage as achieved by increasing the UV dosage in all the phases of the cell cycle. For all the dosages, there is a sharp γH2AX peak in the S phase. (F) Increase in DNA damage always leads to decrease in EU as seen across the population treated with different dosages of UV. But within a population there is no dip in EU levels corresponding to the γH2AX peak showing that γH2AX in the S phase after UV does not reflect on the total extent of DNA damage in those cells. Cells were treated with 10 J/m 2 and 1.6 µg/ml NCS for two minutes. Line graphs are showing relative levels with respect to the mean value of G1 phase in control cells. Scale bar: 10 µm.

    Article Snippet: For measuring global transcription, cells were labelled with 5-Ethynyl-Uridine (EU) immediately after UV irradiation for 30 minutes.

    Techniques:

    RHS are involved in global mRNA synthesis ( A ) Quantification of 5-ethynyl uridine (5-EU) incorporation. Each RNAi line was cultured in the presence or absence of tetracycline for 2 days and pulsed for 10 min with 5-EU ( n = 3). Values are given as a percentage of the uninduced control. P-values are shown for paired, one-tailed t-tests. Error bars = SD. ( B ) Correlation between reads per million (RPM) for nascent mRNAs from RNAi lines cultured in the presence or absence of tetracycline for 2 days. R: Pearson correlation coefficient. Biological replicates were performed. See also Supplemental Figure S5 .

    Journal: Nucleic Acids Research

    Article Title: Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei

    doi: 10.1093/nar/gky1255

    Figure Lengend Snippet: RHS are involved in global mRNA synthesis ( A ) Quantification of 5-ethynyl uridine (5-EU) incorporation. Each RNAi line was cultured in the presence or absence of tetracycline for 2 days and pulsed for 10 min with 5-EU ( n = 3). Values are given as a percentage of the uninduced control. P-values are shown for paired, one-tailed t-tests. Error bars = SD. ( B ) Correlation between reads per million (RPM) for nascent mRNAs from RNAi lines cultured in the presence or absence of tetracycline for 2 days. R: Pearson correlation coefficient. Biological replicates were performed. See also Supplemental Figure S5 .

    Article Snippet: 5-Ethynyl uridine incorporation and processing for GRO-Seq Procyclic forms at a density of 7–8 × 106 ml−1 were pulsed for 10 min with 200 μM 5-ethynyl uridine (5-EU; Jena Biosciences).

    Techniques: Cell Culture, One-tailed Test

    1,6-HD compromises LLPS in living human cells. ( A ) HeLa cells were untreated or treated with Tween 20 (1%, 10 min; “Tween”), Tween 20 followed with 1,6-HD (5%, 15 min; “1,6-HD”) before being analyzed using CellEvent Caspase 3/7 Detection Reagent. The cells treated as described and then incubated in fresh culture medium for 1.5 h were analysed as well. Percent of caspase 3/7-positive (apoptotic) cells is shown. ( B ) HeLa cells treated as described in ( A ) were pulsed with 5-ethyniluridine (EU, 200 μM, 15 min). Box plots show the EU fluorescence intensities. Horizontal lines represent the medians. ( C ) HeLa cells transfected with pHR-FUSN-mCh-Cry2WT were transiently permeabilized and then either mock-treated (control), treated with 1,6-HD (5%, 15 min), or treated with 1,6-HD and allowed to recover for 1.5 h. OptoDroplet formation was monitored as described in ( Shin et al. 2017 ). ( D ) Transiently permeabilized HeLa cells were untreated (control), treated with 1,6-HD (5%, 15 min), or treated with 1,6-HD and allowed to recover for 1.5 h before being stained for coilin (red).

    Journal: bioRxiv

    Article Title: Suppression of liquid-liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells

    doi: 10.1101/2020.05.18.101261

    Figure Lengend Snippet: 1,6-HD compromises LLPS in living human cells. ( A ) HeLa cells were untreated or treated with Tween 20 (1%, 10 min; “Tween”), Tween 20 followed with 1,6-HD (5%, 15 min; “1,6-HD”) before being analyzed using CellEvent Caspase 3/7 Detection Reagent. The cells treated as described and then incubated in fresh culture medium for 1.5 h were analysed as well. Percent of caspase 3/7-positive (apoptotic) cells is shown. ( B ) HeLa cells treated as described in ( A ) were pulsed with 5-ethyniluridine (EU, 200 μM, 15 min). Box plots show the EU fluorescence intensities. Horizontal lines represent the medians. ( C ) HeLa cells transfected with pHR-FUSN-mCh-Cry2WT were transiently permeabilized and then either mock-treated (control), treated with 1,6-HD (5%, 15 min), or treated with 1,6-HD and allowed to recover for 1.5 h. OptoDroplet formation was monitored as described in ( Shin et al. 2017 ). ( D ) Transiently permeabilized HeLa cells were untreated (control), treated with 1,6-HD (5%, 15 min), or treated with 1,6-HD and allowed to recover for 1.5 h before being stained for coilin (red).

    Article Snippet: Measurement of transcriptional activity For 5-ethyniluridine (EU) incorporation, cells were incubated with 200 μM EU (Jena Bioscience) for 15 min at 37°C.

    Techniques: Incubation, Fluorescence, Transfection, Staining