etoposide  (Abcam)

 
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    Name:
    Etoposide
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    ab120227
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    Structured Review

    Abcam etoposide
    DNA damage induces de-phosphorylation of nuclear Tau. ( a ) The effect of <t>Etoposide</t> and Vinblastine treatment on mouse C17.2 cells is shown by confocal microscopy upon immune staining of PFA-fixed cells with antibodies against the microtubule marker β-tubulin (in cyan) or the DNA damage marker γH2A-X (in red). ( b ) Confocal microscopic quantification of the activated kinases in the nucleus (DAPI mask). Mean percent ± sem relative to the respective controls. 2-tailed unpaired Mann-Whitney test, ****p

    https://www.bioz.com/result/etoposide/product/Abcam
    Average 94 stars, based on 2 article reviews
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    etoposide - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Phosphorylation of nuclear Tau is modulated by distinct cellular pathways"

    Article Title: Phosphorylation of nuclear Tau is modulated by distinct cellular pathways

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36374-4

    DNA damage induces de-phosphorylation of nuclear Tau. ( a ) The effect of Etoposide and Vinblastine treatment on mouse C17.2 cells is shown by confocal microscopy upon immune staining of PFA-fixed cells with antibodies against the microtubule marker β-tubulin (in cyan) or the DNA damage marker γH2A-X (in red). ( b ) Confocal microscopic quantification of the activated kinases in the nucleus (DAPI mask). Mean percent ± sem relative to the respective controls. 2-tailed unpaired Mann-Whitney test, ****p
    Figure Legend Snippet: DNA damage induces de-phosphorylation of nuclear Tau. ( a ) The effect of Etoposide and Vinblastine treatment on mouse C17.2 cells is shown by confocal microscopy upon immune staining of PFA-fixed cells with antibodies against the microtubule marker β-tubulin (in cyan) or the DNA damage marker γH2A-X (in red). ( b ) Confocal microscopic quantification of the activated kinases in the nucleus (DAPI mask). Mean percent ± sem relative to the respective controls. 2-tailed unpaired Mann-Whitney test, ****p

    Techniques Used: De-Phosphorylation Assay, Confocal Microscopy, Staining, Marker, MANN-WHITNEY

    2) Product Images from "Phosphorylation of nuclear Tau is modulated by distinct cellular pathways"

    Article Title: Phosphorylation of nuclear Tau is modulated by distinct cellular pathways

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36374-4

    DNA damage induces de-phosphorylation of nuclear Tau. ( a ) The effect of Etoposide and Vinblastine treatment on mouse C17.2 cells is shown by confocal microscopy upon immune staining of PFA-fixed cells with antibodies against the microtubule marker β-tubulin (in cyan) or the DNA damage marker γH2A-X (in red). ( b ) Confocal microscopic quantification of the activated kinases in the nucleus (DAPI mask). Mean percent ± sem relative to the respective controls. 2-tailed unpaired Mann-Whitney test, ****p
    Figure Legend Snippet: DNA damage induces de-phosphorylation of nuclear Tau. ( a ) The effect of Etoposide and Vinblastine treatment on mouse C17.2 cells is shown by confocal microscopy upon immune staining of PFA-fixed cells with antibodies against the microtubule marker β-tubulin (in cyan) or the DNA damage marker γH2A-X (in red). ( b ) Confocal microscopic quantification of the activated kinases in the nucleus (DAPI mask). Mean percent ± sem relative to the respective controls. 2-tailed unpaired Mann-Whitney test, ****p

    Techniques Used: De-Phosphorylation Assay, Confocal Microscopy, Staining, Marker, MANN-WHITNEY

    3) Product Images from "Tau affects P53 function and cell fate during the DNA damage response"

    Article Title: Tau affects P53 function and cell fate during the DNA damage response

    Journal: Communications Biology

    doi: 10.1038/s42003-020-0975-4

    Tau depletion decreases P53 level and apoptosis. For all panels the indicated cell lines (Tau-KO are 232P cells) were treated 30 min with the indicated etoposide concentrations and recovery times. a Mean intensity ± sem of single-cell nuclear P53 staining (DAPI mask, ImageJ) is shown as fold of wt cells at basal conditions, n > 100 cells/condition distributed over five images. b Percent clCasp3-positive cells are shown as mean ± SD of five images for the untreated cells and of 15 images for etoposide-treated cells, n > 500 cells/condition. c Mean intensity ± sem of single-cell Tau staining (tubulin mask, ImageJ) for the indicated cell lines is shown as percent of mock shRNA cells (ctrl), n > 160 cells/condition distributed over five images. d Mean intensity of single-cell nuclear P53 staining quantified as in a , n > 380 cell/condition distributed over 15 images from n = 3 biological replicates. e Percent clCasp3-positive cells quantified as in b , n > 500 cell/condition. f An arbitrary threshold was applied in order to count P53-positive cells as percentage ± SD of total DAPI-positive cell number, n > 100 cells/conditions. For the comparison between the four cell lines ( a ), non-parametric independent Mann–Whitney U test for genotype (in bold) and Kruskal–Wallis pairwise comparison of treatment for cell lines with same genotype (in italics) or for each line (in vertical). For the dose-dependency ( a , b ), non-parametric independent Mann–Whitney U test between cell lines (in bold), Kruskal–Wallis pairwise comparison for each dose (in italics) and between doses (in vertical). Non-parametric independent Mann–Whitney U test ( c – e ) between control and the three Tau-KD lines (in bold) and Kruskal–Wallis pairwise comparison for each Tau-KD line (in italic) and for each treatment (in vertical). Unpaired two-tailed t test with Welch’s correction ( f ).
    Figure Legend Snippet: Tau depletion decreases P53 level and apoptosis. For all panels the indicated cell lines (Tau-KO are 232P cells) were treated 30 min with the indicated etoposide concentrations and recovery times. a Mean intensity ± sem of single-cell nuclear P53 staining (DAPI mask, ImageJ) is shown as fold of wt cells at basal conditions, n > 100 cells/condition distributed over five images. b Percent clCasp3-positive cells are shown as mean ± SD of five images for the untreated cells and of 15 images for etoposide-treated cells, n > 500 cells/condition. c Mean intensity ± sem of single-cell Tau staining (tubulin mask, ImageJ) for the indicated cell lines is shown as percent of mock shRNA cells (ctrl), n > 160 cells/condition distributed over five images. d Mean intensity of single-cell nuclear P53 staining quantified as in a , n > 380 cell/condition distributed over 15 images from n = 3 biological replicates. e Percent clCasp3-positive cells quantified as in b , n > 500 cell/condition. f An arbitrary threshold was applied in order to count P53-positive cells as percentage ± SD of total DAPI-positive cell number, n > 100 cells/conditions. For the comparison between the four cell lines ( a ), non-parametric independent Mann–Whitney U test for genotype (in bold) and Kruskal–Wallis pairwise comparison of treatment for cell lines with same genotype (in italics) or for each line (in vertical). For the dose-dependency ( a , b ), non-parametric independent Mann–Whitney U test between cell lines (in bold), Kruskal–Wallis pairwise comparison for each dose (in italics) and between doses (in vertical). Non-parametric independent Mann–Whitney U test ( c – e ) between control and the three Tau-KD lines (in bold) and Kruskal–Wallis pairwise comparison for each Tau-KD line (in italic) and for each treatment (in vertical). Unpaired two-tailed t test with Welch’s correction ( f ).

    Techniques Used: Staining, shRNA, MANN-WHITNEY, Two Tailed Test

    Tau does not directly interact with P53 . Cell lysates of SH-SY5Y treated with 10 μM of MG132 to stabilize P53 expression, without (ctrl) or with (eto) a 30 min pre-treatment with 60 μM etoposide, were subjected to immune precipitation of endogenous P53 with a rabbit antibody (P53) or with a rabbit GFP antibody as negative IP control (GFP). Western blot analysis for co-precipitation of MDM2 or Tau with the respective mouse antibodies as indicated. The blots on the top show the analysis of the starting material (cell lysates), those on the bottom the immunoprecipitation (IP). The P53 blots are entirely shown, whereas for MDM2 and Tau, the blots were cut between the 55 kDa and the 95 kDa protein size markers and analyzed separately.
    Figure Legend Snippet: Tau does not directly interact with P53 . Cell lysates of SH-SY5Y treated with 10 μM of MG132 to stabilize P53 expression, without (ctrl) or with (eto) a 30 min pre-treatment with 60 μM etoposide, were subjected to immune precipitation of endogenous P53 with a rabbit antibody (P53) or with a rabbit GFP antibody as negative IP control (GFP). Western blot analysis for co-precipitation of MDM2 or Tau with the respective mouse antibodies as indicated. The blots on the top show the analysis of the starting material (cell lysates), those on the bottom the immunoprecipitation (IP). The P53 blots are entirely shown, whereas for MDM2 and Tau, the blots were cut between the 55 kDa and the 95 kDa protein size markers and analyzed separately.

    Techniques Used: Expressing, Western Blot, Immunoprecipitation

    Differential regulation of P53 transcription targets . Extracted RNA from parental (wt) and 232P (Tau-KO) cells in a or from control shRNA plasmid (ctrl) and 3127 (Tau-KD) cells in b at basal conditions or after 30 min 60 μM etoposide and 6 h recovery, was subjected to reverse-transcription and qPCR with primers specific for the indicated transcripts. Mean ± SD of relative mRNA levels ( n = 3) shown as fold of the respective basal conditions for parental or control cells. Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (horizontal), multiple Sidak pairwise comparisons for treatment for each line (in vertical).
    Figure Legend Snippet: Differential regulation of P53 transcription targets . Extracted RNA from parental (wt) and 232P (Tau-KO) cells in a or from control shRNA plasmid (ctrl) and 3127 (Tau-KD) cells in b at basal conditions or after 30 min 60 μM etoposide and 6 h recovery, was subjected to reverse-transcription and qPCR with primers specific for the indicated transcripts. Mean ± SD of relative mRNA levels ( n = 3) shown as fold of the respective basal conditions for parental or control cells. Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (horizontal), multiple Sidak pairwise comparisons for treatment for each line (in vertical).

    Techniques Used: shRNA, Plasmid Preparation, Real-time Polymerase Chain Reaction

    Role of P53 and MDM2 modifications for P53 function and stability . a , b Parental (wt) or 232P (Tau-KO) cells treated 30 min without (basal) or with 60 μM etoposide and recovered for 6 h in the absence (eto) or presence of 10 µg/mL KU-55933 and/or 5 µg/mL nutlin-3. a Mean intensity ± sem of single-cell nuclear P53 or MDM2 (DAPI mask, ImageJ) shown as fold of basal conditions, n > 100 cells/condition distributed over five images. b Percent clCasp3-positive cells shown as mean ± SD of five images (basal) or 15 images (treatments), n > 500 cells/condition. Non-parametric independent samples test and Kruskal–Wallis pairwise comparison between cell lines (in bold) or for treatment for each cell line (in vertical). c Western bot analysis of P53 in parental (wt) or 232 P (Tau-KO) cells at basal conditions, after 30 min 60 μM etoposide and 4 h recovery without or with 10 μM MG132. GAPDH served as loading control. d Parental (wt) or 232P (Tau-KO) cells pre-treated for 30 min with 60 μM etoposide followed by 4 h with 10 μM MG132, were incubated with 25 μM of cycloheximide (CHX) for the indicated chase times. Single-cell nuclear P53 or nuclear MDM2 (DAPI mask, ImageJ) shown as fold of wt cells at basal conditions. Mean intensity ± sem of n > 100 cells/condition distributed over five images. Independent measures ordinary two-way ANOVA, source of variation for cell lines (bold), multiple Bonferroni pairwise comparisons of treatment for each line (in italic). e Parental (wt) or (Tau-KO) 232P cells treated for 30 min with 60 μM etoposide and 6 h recovery analyzed with a 90 kDa MDM2 rabbit antibody (green and middle panel with GAPDH as loading control) or a 60 and 90 kDa MDM2 mouse antibody (red and bottom panel).
    Figure Legend Snippet: Role of P53 and MDM2 modifications for P53 function and stability . a , b Parental (wt) or 232P (Tau-KO) cells treated 30 min without (basal) or with 60 μM etoposide and recovered for 6 h in the absence (eto) or presence of 10 µg/mL KU-55933 and/or 5 µg/mL nutlin-3. a Mean intensity ± sem of single-cell nuclear P53 or MDM2 (DAPI mask, ImageJ) shown as fold of basal conditions, n > 100 cells/condition distributed over five images. b Percent clCasp3-positive cells shown as mean ± SD of five images (basal) or 15 images (treatments), n > 500 cells/condition. Non-parametric independent samples test and Kruskal–Wallis pairwise comparison between cell lines (in bold) or for treatment for each cell line (in vertical). c Western bot analysis of P53 in parental (wt) or 232 P (Tau-KO) cells at basal conditions, after 30 min 60 μM etoposide and 4 h recovery without or with 10 μM MG132. GAPDH served as loading control. d Parental (wt) or 232P (Tau-KO) cells pre-treated for 30 min with 60 μM etoposide followed by 4 h with 10 μM MG132, were incubated with 25 μM of cycloheximide (CHX) for the indicated chase times. Single-cell nuclear P53 or nuclear MDM2 (DAPI mask, ImageJ) shown as fold of wt cells at basal conditions. Mean intensity ± sem of n > 100 cells/condition distributed over five images. Independent measures ordinary two-way ANOVA, source of variation for cell lines (bold), multiple Bonferroni pairwise comparisons of treatment for each line (in italic). e Parental (wt) or (Tau-KO) 232P cells treated for 30 min with 60 μM etoposide and 6 h recovery analyzed with a 90 kDa MDM2 rabbit antibody (green and middle panel with GAPDH as loading control) or a 60 and 90 kDa MDM2 mouse antibody (red and bottom panel).

    Techniques Used: Western Blot, Incubation

    Tau deficiency confers resistance to etoposide-induced apoptosis . Scheme representing the design of the experiment with parental and 231A (Tau) cells compared to 232P and 231K (Tau-KO) cells treated 30 min with 60 μM etoposide and recovered as indicated before analysis. LDH and MTS values are shown as percentage of parental cells (wt), mean ± SD of five biological replicates. To measure activation of apoptosis, percent positive cells for cleaved-caspase-3 (clCasp3) is determined on confocal microscope images and normalized for total DAPI-positive cells, mean ± SD of five images for the untreated cells (ctrl) and of 15 images for etoposide-treated cells (60 μM eto), n > 500 cells/condition, representative experiment of n > 3 biological replicates. Activated clCasp3 was also analyzed by western blot with GAPDH as loading control and 15 and 17 kDa clCasp3 quantified by normalization with GAPDH, mean ± SD ( n = 3 biological triplicates). Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (in bold), multiple Bonferroni pairwise comparisons for treatment between lines (in italics) or for each line (in vertical).
    Figure Legend Snippet: Tau deficiency confers resistance to etoposide-induced apoptosis . Scheme representing the design of the experiment with parental and 231A (Tau) cells compared to 232P and 231K (Tau-KO) cells treated 30 min with 60 μM etoposide and recovered as indicated before analysis. LDH and MTS values are shown as percentage of parental cells (wt), mean ± SD of five biological replicates. To measure activation of apoptosis, percent positive cells for cleaved-caspase-3 (clCasp3) is determined on confocal microscope images and normalized for total DAPI-positive cells, mean ± SD of five images for the untreated cells (ctrl) and of 15 images for etoposide-treated cells (60 μM eto), n > 500 cells/condition, representative experiment of n > 3 biological replicates. Activated clCasp3 was also analyzed by western blot with GAPDH as loading control and 15 and 17 kDa clCasp3 quantified by normalization with GAPDH, mean ± SD ( n = 3 biological triplicates). Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (in bold), multiple Bonferroni pairwise comparisons for treatment between lines (in italics) or for each line (in vertical).

    Techniques Used: Activation Assay, Microscopy, Western Blot

    Reduced P53 in Tau-KO cells is not caused by DDR activation . For all panels, parental (wt) or 232P (Tau-KO) cells were treated 30 min with the indicated etoposide concentrations and recovery times. a Mean intensity ± SD of γH2A-X staining normalized for DAPI staining by in-cell western is shown as fold of wt cells at basal conditions, n = 5 of biological replicates in a 96-well plate. Non-parametric independent Mann–Whitney U test between lines (in bold), or for each dose between lines (in italics). b Olive moment in the Comet assay is shown as mean ± sem, n = 84–146 cells/condition. c , d Mean intensity ± sem of single-cell nuclear γH2A-X, pATM or pChk2 staining (DAPI mask, ImageJ) is shown as fold of wt cells at basal conditions, n > 100 cells/condition distributed over five images. Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (in bold), multiple Bonferroni pairwise comparisons of each condition between lines (in italics) and of time points for each line ( b , d , in vertical).
    Figure Legend Snippet: Reduced P53 in Tau-KO cells is not caused by DDR activation . For all panels, parental (wt) or 232P (Tau-KO) cells were treated 30 min with the indicated etoposide concentrations and recovery times. a Mean intensity ± SD of γH2A-X staining normalized for DAPI staining by in-cell western is shown as fold of wt cells at basal conditions, n = 5 of biological replicates in a 96-well plate. Non-parametric independent Mann–Whitney U test between lines (in bold), or for each dose between lines (in italics). b Olive moment in the Comet assay is shown as mean ± sem, n = 84–146 cells/condition. c , d Mean intensity ± sem of single-cell nuclear γH2A-X, pATM or pChk2 staining (DAPI mask, ImageJ) is shown as fold of wt cells at basal conditions, n > 100 cells/condition distributed over five images. Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (in bold), multiple Bonferroni pairwise comparisons of each condition between lines (in italics) and of time points for each line ( b , d , in vertical).

    Techniques Used: Activation Assay, Staining, In-Cell ELISA, MANN-WHITNEY, Single Cell Gel Electrophoresis

    Tau depletion increases cellular senescence . a Scheme of the procedure followed to assess cellular senescence upon 30 min treatment with 60 μM etoposide followed by 3 days of recovery. b Quantification of p21 amount in cell lysates by western blot in parental (wt) or 232P (Tau-KO) cells under control conditions (ctrl) or following etoposide treatment (60 µM eto) normalized for GAPDH, mean ± SD of three biological replicates. Quantification of mean cell area and percent positive cells for senescence-associated β-galactosidase (SA- βGal) determined with a high-content microscope scanner, mean ± sem of four (Tau-KO cells) or three (Tau-KD) independent experiments, n > 8000 cells. Data are shown as fold of wt cells at basal conditions. c Same as in b for mock shRNA (ctrl) or Tau 3127 shRNA (Tau-KD) cells. d Representative images of SA-βGal staining (in blue), bright-field, scale bar = 100 μm. Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (in bold), multiple Bonferroni pairwise comparisons for treatment between lines (in italics) or for each line (in vertical).
    Figure Legend Snippet: Tau depletion increases cellular senescence . a Scheme of the procedure followed to assess cellular senescence upon 30 min treatment with 60 μM etoposide followed by 3 days of recovery. b Quantification of p21 amount in cell lysates by western blot in parental (wt) or 232P (Tau-KO) cells under control conditions (ctrl) or following etoposide treatment (60 µM eto) normalized for GAPDH, mean ± SD of three biological replicates. Quantification of mean cell area and percent positive cells for senescence-associated β-galactosidase (SA- βGal) determined with a high-content microscope scanner, mean ± sem of four (Tau-KO cells) or three (Tau-KD) independent experiments, n > 8000 cells. Data are shown as fold of wt cells at basal conditions. c Same as in b for mock shRNA (ctrl) or Tau 3127 shRNA (Tau-KD) cells. d Representative images of SA-βGal staining (in blue), bright-field, scale bar = 100 μm. Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (in bold), multiple Bonferroni pairwise comparisons for treatment between lines (in italics) or for each line (in vertical).

    Techniques Used: Western Blot, Microscopy, shRNA, Staining

    4) Product Images from "Feasibility, potency, and safety of growing human mesenchymal stem cells in space for clinical application"

    Article Title: Feasibility, potency, and safety of growing human mesenchymal stem cells in space for clinical application

    Journal: NPJ Microgravity

    doi: 10.1038/s41526-020-0106-z

    Genomic integrity analysis of the safety of growing MSC in space. a Bone marrow-derived mesenchymal stem cells (5000 cells/well) previously grown for 1 week or 2 weeks in space, along with ground control cells were seeded to ibidi µ-Slide 8 Well. Cells were stimulated with 100 μM etoposide as indicated, fixed, and subjected to immunofluorescence analysis to visualize pS139-H2A.X. Scale bars indicate 10 μm. b shows quantitation of the fluorescence intensity of pS139-H2A.X ( n = 40 cells). Analysis was conducted using the Image J software. Fluorescence intensities were normalized to the etoposide-treated group for each experimental condition. **** indicates statistical significance (
    Figure Legend Snippet: Genomic integrity analysis of the safety of growing MSC in space. a Bone marrow-derived mesenchymal stem cells (5000 cells/well) previously grown for 1 week or 2 weeks in space, along with ground control cells were seeded to ibidi µ-Slide 8 Well. Cells were stimulated with 100 μM etoposide as indicated, fixed, and subjected to immunofluorescence analysis to visualize pS139-H2A.X. Scale bars indicate 10 μm. b shows quantitation of the fluorescence intensity of pS139-H2A.X ( n = 40 cells). Analysis was conducted using the Image J software. Fluorescence intensities were normalized to the etoposide-treated group for each experimental condition. **** indicates statistical significance (

    Techniques Used: Derivative Assay, Immunofluorescence, Quantitation Assay, Fluorescence, Software

    5) Product Images from "DNA damage induces a meiotic arrest in mouse oocytes mediated by the spindle assembly checkpoint"

    Article Title: DNA damage induces a meiotic arrest in mouse oocytes mediated by the spindle assembly checkpoint

    Journal: Nature Communications

    doi: 10.1038/ncomms9553

    DNA damage following NEB causes MI arrest. ( a – c ) MI-arrest rates in oocytes that followed DNA damage induction either during GV arrest or 1 h after NEB; ( a ) etoposide addition (25 μg ml −1 ), ( b ) bleomycin (1 μM); and ( c ) ultraviolet B exposure (300 nm, 30 s). Numbers of oocytes used are as indicated (pooled from between 2 and 6 mice per treatment); ( a , b ) compared against vehicle addition; ( c ) no exposure. * P
    Figure Legend Snippet: DNA damage following NEB causes MI arrest. ( a – c ) MI-arrest rates in oocytes that followed DNA damage induction either during GV arrest or 1 h after NEB; ( a ) etoposide addition (25 μg ml −1 ), ( b ) bleomycin (1 μM); and ( c ) ultraviolet B exposure (300 nm, 30 s). Numbers of oocytes used are as indicated (pooled from between 2 and 6 mice per treatment); ( a , b ) compared against vehicle addition; ( c ) no exposure. * P

    Techniques Used: Mouse Assay

    DNA damage during GV arrest induces an MI arrest. ( a ) PBE in untreated oocytes at the times indicated after NEB. The majority of oocytes complete MI between 8 and 11 h after NEB. Data are from 59 oocytes, pooled from three mice. Images of PBE, scale bar, 50 μm. ( b ) Schematic of the experimental design to assess the effects of DNA damage on meiotic progression. ( c – f ) MI-arrest rates in response to DNA damage that was induced during GV arrest with various doses of etoposide ( c ) bleomycin ( d ) ultraviolet B (UVB) exposure at 300 nm ( e ) or ionizing radiation (IR) ( f ). Arrest was assessed at 15 h. ( g ) Prolonged MI arrest rate at 25 h after NEB. Oocytes that were initially MI arrested at 15 h, following GV-stage DNA damage (etoposide 25 μg ml −1 , bleomycin 10 μM, ultraviolet B exposure 15 s, ionizing radiation 12.3 Gy), were further cultured for 10 h and assessed for PBE. ( c – g ) Numbers of oocytes used are as indicated (pooled from between two and six mice per treatment). * P
    Figure Legend Snippet: DNA damage during GV arrest induces an MI arrest. ( a ) PBE in untreated oocytes at the times indicated after NEB. The majority of oocytes complete MI between 8 and 11 h after NEB. Data are from 59 oocytes, pooled from three mice. Images of PBE, scale bar, 50 μm. ( b ) Schematic of the experimental design to assess the effects of DNA damage on meiotic progression. ( c – f ) MI-arrest rates in response to DNA damage that was induced during GV arrest with various doses of etoposide ( c ) bleomycin ( d ) ultraviolet B (UVB) exposure at 300 nm ( e ) or ionizing radiation (IR) ( f ). Arrest was assessed at 15 h. ( g ) Prolonged MI arrest rate at 25 h after NEB. Oocytes that were initially MI arrested at 15 h, following GV-stage DNA damage (etoposide 25 μg ml −1 , bleomycin 10 μM, ultraviolet B exposure 15 s, ionizing radiation 12.3 Gy), were further cultured for 10 h and assessed for PBE. ( c – g ) Numbers of oocytes used are as indicated (pooled from between two and six mice per treatment). * P

    Techniques Used: Mouse Assay, Cell Culture

    DNA damage permits spindle formation but causes DNA fragmentation. ( a ) Meiotic spindle measurements of length and width taken on fixed oocytes, following addition of either vehicle or etoposide for 15 min during GV arrest. Neither spindle length nor width was affected by etoposide addition (n.s., P > 0.05, ANOVA with Tukey's post hoc analysis). Between 9 and 45 oocytes were analysed per group, with oocytes pooled from six mice, displayed are means with s.d. ( b ) A chromosome fragment (Fr) generated in an oocyte following etoposide addition (25 μg ml −1 ). This fragment possesses a single kinetochore pair, seen by counting kinetochores in the individual z -slices (numbered 1–8). For comparison, the two sister kinetochore pairs of a bivalent are also illustrated (Bv). ( c ) Percentage of etoposide- (25 μg ml −1 ) or ultraviolet B (300 nm, 15 s)-treated oocytes, expressing CenpC and H2B, found to have bivalent fragments during 4D CLSM. Images were captured every 10 min over a period of 15 h. Numbers of oocytes used are indicated (data pooled from two mice for each condition). The rate of fragmentation was precisely measured in each oocyte, and not under-reported, by counting for the expected number of kinetochores (40 CenpC foci) and ensuring that no DNA fragments were outside of the imaging volume. * P
    Figure Legend Snippet: DNA damage permits spindle formation but causes DNA fragmentation. ( a ) Meiotic spindle measurements of length and width taken on fixed oocytes, following addition of either vehicle or etoposide for 15 min during GV arrest. Neither spindle length nor width was affected by etoposide addition (n.s., P > 0.05, ANOVA with Tukey's post hoc analysis). Between 9 and 45 oocytes were analysed per group, with oocytes pooled from six mice, displayed are means with s.d. ( b ) A chromosome fragment (Fr) generated in an oocyte following etoposide addition (25 μg ml −1 ). This fragment possesses a single kinetochore pair, seen by counting kinetochores in the individual z -slices (numbered 1–8). For comparison, the two sister kinetochore pairs of a bivalent are also illustrated (Bv). ( c ) Percentage of etoposide- (25 μg ml −1 ) or ultraviolet B (300 nm, 15 s)-treated oocytes, expressing CenpC and H2B, found to have bivalent fragments during 4D CLSM. Images were captured every 10 min over a period of 15 h. Numbers of oocytes used are indicated (data pooled from two mice for each condition). The rate of fragmentation was precisely measured in each oocyte, and not under-reported, by counting for the expected number of kinetochores (40 CenpC foci) and ensuring that no DNA fragments were outside of the imaging volume. * P

    Techniques Used: Mouse Assay, Generated, Expressing, Confocal Laser Scanning Microscopy, Imaging

    MI arrest following DNA damage is independent of bivalent biorientation. ( a ) Percentage of oocytes that undergo PBE or MI arrest following GV-stage treatment with ultraviolet B (15 s), etoposide (25 μg ml −1 , 15 min), or a low dose of nocodazole (25 nM) added for the duration of MI. ( b – d ) Bivalent tension (stretch), displacement from the metaphase plate (displacement) and bivalent orientation ( θ ); in oocytes treated throughout MI with nocodazole ( b ) or before NEB for 15 s with ultraviolet B ( c ) or 15 min with etoposide ( d ). Data points are individual bivalents combined from three oocytes per treatment group at 8 h. The maximum number of s.d.'s from the 8 h PBE group mean on any axis is used to colour the bivalent. The three nocodazole-treated oocytes all underwent anaphase in the next 10 min. ( e – g ) Bivalent stretch ( e ) displacement ( e ) and orientation ( f ) from untreated oocytes and those in ( b – d ). Each group assessed 3 oocytes and 60 bivalents. Bars indicate means, and errors are s.d. Background colouring represents multiple s.d.'s from the mean in the control group. *, indicates significant difference from control, P
    Figure Legend Snippet: MI arrest following DNA damage is independent of bivalent biorientation. ( a ) Percentage of oocytes that undergo PBE or MI arrest following GV-stage treatment with ultraviolet B (15 s), etoposide (25 μg ml −1 , 15 min), or a low dose of nocodazole (25 nM) added for the duration of MI. ( b – d ) Bivalent tension (stretch), displacement from the metaphase plate (displacement) and bivalent orientation ( θ ); in oocytes treated throughout MI with nocodazole ( b ) or before NEB for 15 s with ultraviolet B ( c ) or 15 min with etoposide ( d ). Data points are individual bivalents combined from three oocytes per treatment group at 8 h. The maximum number of s.d.'s from the 8 h PBE group mean on any axis is used to colour the bivalent. The three nocodazole-treated oocytes all underwent anaphase in the next 10 min. ( e – g ) Bivalent stretch ( e ) displacement ( e ) and orientation ( f ) from untreated oocytes and those in ( b – d ). Each group assessed 3 oocytes and 60 bivalents. Bars indicate means, and errors are s.d. Background colouring represents multiple s.d.'s from the mean in the control group. *, indicates significant difference from control, P

    Techniques Used:

    DNA damage induction and repair in GV oocytes. ( a ) Representative γH2AX immunofluorescence in a GV oocyte following either no treatment (control), or etoposide (25 μg ml −1 ). γH2AX staining is negligible in the control oocyte but visible in the nucleus following DNA damage. Scale bar, 5 μm. ( b ) Nuclear γH2AX immunofluorescence levels in individual oocytes following etopiside (25 μg ml −1 ), bleomycin (1 μM), ultraviolet B (UVB, 300 nm, 30 s) or ionizing radiation (IR, 4.5 Gy). Data are pooled from three mice for each condition. ( c ) Nuclear γH2AX accumulation in oocytes at various times after etoposide (25 μg ml −1 ) exposure. Oocytes were maintained in GV arrest for the times indicated after 15-min exposure to etoposide or vehicle (0.1% DMSO). Nuclear γH2AX accumulation following etoposide exposure is initially high, but drops to control levels over 10 h. Oocytes were pooled from four mice. ( a – c ) Oocytes were fixed at either 15 min (etoposide and bleomycin) or 40 min after treatment. ( b , c ) Each data point represents one oocyte; means and s.d. are represented by the horizontal lines; * P
    Figure Legend Snippet: DNA damage induction and repair in GV oocytes. ( a ) Representative γH2AX immunofluorescence in a GV oocyte following either no treatment (control), or etoposide (25 μg ml −1 ). γH2AX staining is negligible in the control oocyte but visible in the nucleus following DNA damage. Scale bar, 5 μm. ( b ) Nuclear γH2AX immunofluorescence levels in individual oocytes following etopiside (25 μg ml −1 ), bleomycin (1 μM), ultraviolet B (UVB, 300 nm, 30 s) or ionizing radiation (IR, 4.5 Gy). Data are pooled from three mice for each condition. ( c ) Nuclear γH2AX accumulation in oocytes at various times after etoposide (25 μg ml −1 ) exposure. Oocytes were maintained in GV arrest for the times indicated after 15-min exposure to etoposide or vehicle (0.1% DMSO). Nuclear γH2AX accumulation following etoposide exposure is initially high, but drops to control levels over 10 h. Oocytes were pooled from four mice. ( a – c ) Oocytes were fixed at either 15 min (etoposide and bleomycin) or 40 min after treatment. ( b , c ) Each data point represents one oocyte; means and s.d. are represented by the horizontal lines; * P

    Techniques Used: Immunofluorescence, Staining, Mouse Assay

    Loss of Mps1 activity overcomes DNA-damage-induced MI arrest. ( a ) MI completion rates following addition of the Mps1 inhibitor reversine (100 nM) to oocytes, at 11 h after NEB, that had been arrested in MI through various methods of DNA damage (etoposide 25 μg ml −1 ; bleomycin 1 μM; ultraviolet B, 15 s; ionizing radiation, 1.2 Gy). Numbers of oocytes used are indicated (data pooled from three to four mice). As a control, vehicle was added to bleomycin-treated oocytes, and all of these remained MI arrested. ( b ) Cumulative MI completion rates for oocytes treated with bleomycin (1 μM), ultraviolet B (300 nm, 15 s) or vehicle (0.1% DMSO) before NEB, and then cultured in the presence of 100 nM reversine (solid lines), or control oocytes without any treatment (dashed line). ( c ) Securin-YFP degradation measured relative to fluorescence at 3 h, in either non-DNA damaged or DNA damaged (etoposide 25 μg ml −1 for 15 min during GV arrest). Individual oocytes are recorded, pooled from at least two mice.
    Figure Legend Snippet: Loss of Mps1 activity overcomes DNA-damage-induced MI arrest. ( a ) MI completion rates following addition of the Mps1 inhibitor reversine (100 nM) to oocytes, at 11 h after NEB, that had been arrested in MI through various methods of DNA damage (etoposide 25 μg ml −1 ; bleomycin 1 μM; ultraviolet B, 15 s; ionizing radiation, 1.2 Gy). Numbers of oocytes used are indicated (data pooled from three to four mice). As a control, vehicle was added to bleomycin-treated oocytes, and all of these remained MI arrested. ( b ) Cumulative MI completion rates for oocytes treated with bleomycin (1 μM), ultraviolet B (300 nm, 15 s) or vehicle (0.1% DMSO) before NEB, and then cultured in the presence of 100 nM reversine (solid lines), or control oocytes without any treatment (dashed line). ( c ) Securin-YFP degradation measured relative to fluorescence at 3 h, in either non-DNA damaged or DNA damaged (etoposide 25 μg ml −1 for 15 min during GV arrest). Individual oocytes are recorded, pooled from at least two mice.

    Techniques Used: Activity Assay, Mouse Assay, Cell Culture, Fluorescence

    6) Product Images from "The pro-apoptotic effect of a Terpene-rich Annona cherimola leaf extract on leukemic cell lines"

    Article Title: The pro-apoptotic effect of a Terpene-rich Annona cherimola leaf extract on leukemic cell lines

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/s12906-019-2768-1

    The quantitative effect of AELE on induction of apoptosis using Cell Death ELISA. Cell Death ELISA on Monomac-1 ( a ) and KG-1 ( b ) cells, treated with the two concentrations of AELE closest to the IC50 (173 and 346 μg/mL), as well as a positive control treated with etoposide for 24 h. A significant dose-dependent increase in enrichment factor is noted for AML cells upon treatment with two increasing doses of AELE for 24 h. (** indicates a p- value: 0.001
    Figure Legend Snippet: The quantitative effect of AELE on induction of apoptosis using Cell Death ELISA. Cell Death ELISA on Monomac-1 ( a ) and KG-1 ( b ) cells, treated with the two concentrations of AELE closest to the IC50 (173 and 346 μg/mL), as well as a positive control treated with etoposide for 24 h. A significant dose-dependent increase in enrichment factor is noted for AML cells upon treatment with two increasing doses of AELE for 24 h. (** indicates a p- value: 0.001

    Techniques Used: Enzyme-linked Immunosorbent Assay, Positive Control

    7) Product Images from "DNA mismatch repair controls the host innate response and cell fate after influenza virus infection"

    Article Title: DNA mismatch repair controls the host innate response and cell fate after influenza virus infection

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0509-3

    DNA MMR activity remains high in H441 cells allowing repair of virally-induced ROS-mediated DNA damage. (a) Representative phospho-H2AX (red) and nuclei (blue) staining of H441 cells to measure the level of DNA damage present during WT PR8 infection. Etoposide and H 2 O 2 are used as positive controls. (b) Quantification of the mean intensity of the phospho-H2AX staining from samples in a. Data shown as mean ± SD, n=3 independent samples. Representative of two independent experiments. (c) Histogram of 8-Oxo-2’-deoxyguanosine (8-OHdG) in H441 cells comparing mock-infected to PR8-infected at 48 hpi. Representative of two independent experiments. (d) Quantification of the geometric mean of fluorescence of the 8-OHdG staining from c. Data shown as mean ± SD, n=6 independent samples. Representative of two independent experiments. (e) Representative images of the modified Comet assay used to compare the level of oxidative DNA damage following siRNA knockdown of control or DNA MMR genes MSH2+MSH6 and mock or WT PR8 infection. Longer tails (indicated by white arrows) correspond to greater DNA damage. (f) Quantification of comet tail lengths (normalized to nuclear diameter) in the samples displayed in e. Data shown as mean ± SD, n=50 nuclei. Representative of two independent experiments. (g) Percent of surviving A549-CR cells after treatment with 0.5 mM Trolox or mock control and infection with IAV-Cre for 120 h. Data shown as mean ± SD, n=8 independent samples. Representative of five experiments. (h) Percent of surviving H441-CR cells after treatment with 0.5 mM Trolox or mock control and infection with IAV-Cre for 120 h. Data shown as mean ± SD, n=6 independent samples. Representative of four experiments. For all panels: p-values calculated using unpaired two-tailed t tests; scale bars = 100 μm.
    Figure Legend Snippet: DNA MMR activity remains high in H441 cells allowing repair of virally-induced ROS-mediated DNA damage. (a) Representative phospho-H2AX (red) and nuclei (blue) staining of H441 cells to measure the level of DNA damage present during WT PR8 infection. Etoposide and H 2 O 2 are used as positive controls. (b) Quantification of the mean intensity of the phospho-H2AX staining from samples in a. Data shown as mean ± SD, n=3 independent samples. Representative of two independent experiments. (c) Histogram of 8-Oxo-2’-deoxyguanosine (8-OHdG) in H441 cells comparing mock-infected to PR8-infected at 48 hpi. Representative of two independent experiments. (d) Quantification of the geometric mean of fluorescence of the 8-OHdG staining from c. Data shown as mean ± SD, n=6 independent samples. Representative of two independent experiments. (e) Representative images of the modified Comet assay used to compare the level of oxidative DNA damage following siRNA knockdown of control or DNA MMR genes MSH2+MSH6 and mock or WT PR8 infection. Longer tails (indicated by white arrows) correspond to greater DNA damage. (f) Quantification of comet tail lengths (normalized to nuclear diameter) in the samples displayed in e. Data shown as mean ± SD, n=50 nuclei. Representative of two independent experiments. (g) Percent of surviving A549-CR cells after treatment with 0.5 mM Trolox or mock control and infection with IAV-Cre for 120 h. Data shown as mean ± SD, n=8 independent samples. Representative of five experiments. (h) Percent of surviving H441-CR cells after treatment with 0.5 mM Trolox or mock control and infection with IAV-Cre for 120 h. Data shown as mean ± SD, n=6 independent samples. Representative of four experiments. For all panels: p-values calculated using unpaired two-tailed t tests; scale bars = 100 μm.

    Techniques Used: Activity Assay, Staining, Infection, Fluorescence, Modification, Single Cell Gel Electrophoresis, Two Tailed Test

    Related Articles

    Incubation:

    Article Title: Phosphorylation of nuclear Tau is modulated by distinct cellular pathways
    Article Snippet: .. Drug treatments During the last 5 h of tetracycline incubation, C17.2 cells with inducible Tau expression were treated with 60 µM Etoposide (Abcam, ab120227; 100 mM stock in DMSO), 3 µM Vinblastine (Sigma-Aldrich, V1377; 11 mM stock in DMSO), or 60 nM Leptomycin B (Sigma-Aldrich, L2913; 10.3 µM in 70% ethanol). ..

    Article Title: Phosphorylation of nuclear Tau is modulated by distinct cellular pathways
    Article Snippet: .. During the last 5 h of tetracycline incubation, C17.2 cells with inducible Tau expression were treated with 60 µM Etoposide (Abcam, ab120227; 100 mM stock in DMSO), 3 µM Vinblastine (Sigma-Aldrich, V1377; 11 mM stock in DMSO), or 60 nM Leptomycin B (Sigma-Aldrich, L2913; 10.3 µM in 70% ethanol). ..

    other:

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences
    Article Snippet: Synthesis of the activated etoposide core, shown in the top portion of Figure , started with commercially available DEPT ( 1 ) (ABCAM Biochemicals).

    Expressing:

    Article Title: Phosphorylation of nuclear Tau is modulated by distinct cellular pathways
    Article Snippet: .. Drug treatments During the last 5 h of tetracycline incubation, C17.2 cells with inducible Tau expression were treated with 60 µM Etoposide (Abcam, ab120227; 100 mM stock in DMSO), 3 µM Vinblastine (Sigma-Aldrich, V1377; 11 mM stock in DMSO), or 60 nM Leptomycin B (Sigma-Aldrich, L2913; 10.3 µM in 70% ethanol). ..

    Article Title: Phosphorylation of nuclear Tau is modulated by distinct cellular pathways
    Article Snippet: .. During the last 5 h of tetracycline incubation, C17.2 cells with inducible Tau expression were treated with 60 µM Etoposide (Abcam, ab120227; 100 mM stock in DMSO), 3 µM Vinblastine (Sigma-Aldrich, V1377; 11 mM stock in DMSO), or 60 nM Leptomycin B (Sigma-Aldrich, L2913; 10.3 µM in 70% ethanol). ..

    Positive Control:

    Article Title: The pro-apoptotic effect of a Terpene-rich Annona cherimola leaf extract on leukemic cell lines
    Article Snippet: .. A positive control well, treated with 100 μM of etoposide (Abcam), was also included. .. Cells were extracted and lysed with incubation buffer, using the Cell Death ELISA kit (Roche), before isolation of fragmented cytosolic DNA.

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    Abcam etoposide
    DNA damage induces de-phosphorylation of nuclear Tau. ( a ) The effect of <t>Etoposide</t> and Vinblastine treatment on mouse C17.2 cells is shown by confocal microscopy upon immune staining of PFA-fixed cells with antibodies against the microtubule marker β-tubulin (in cyan) or the DNA damage marker γH2A-X (in red). ( b ) Confocal microscopic quantification of the activated kinases in the nucleus (DAPI mask). Mean percent ± sem relative to the respective controls. 2-tailed unpaired Mann-Whitney test, ****p
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    DNA damage induces de-phosphorylation of nuclear Tau. ( a ) The effect of Etoposide and Vinblastine treatment on mouse C17.2 cells is shown by confocal microscopy upon immune staining of PFA-fixed cells with antibodies against the microtubule marker β-tubulin (in cyan) or the DNA damage marker γH2A-X (in red). ( b ) Confocal microscopic quantification of the activated kinases in the nucleus (DAPI mask). Mean percent ± sem relative to the respective controls. 2-tailed unpaired Mann-Whitney test, ****p

    Journal: Scientific Reports

    Article Title: Phosphorylation of nuclear Tau is modulated by distinct cellular pathways

    doi: 10.1038/s41598-018-36374-4

    Figure Lengend Snippet: DNA damage induces de-phosphorylation of nuclear Tau. ( a ) The effect of Etoposide and Vinblastine treatment on mouse C17.2 cells is shown by confocal microscopy upon immune staining of PFA-fixed cells with antibodies against the microtubule marker β-tubulin (in cyan) or the DNA damage marker γH2A-X (in red). ( b ) Confocal microscopic quantification of the activated kinases in the nucleus (DAPI mask). Mean percent ± sem relative to the respective controls. 2-tailed unpaired Mann-Whitney test, ****p

    Article Snippet: Drug treatments During the last 5 h of tetracycline incubation, C17.2 cells with inducible Tau expression were treated with 60 µM Etoposide (Abcam, ab120227; 100 mM stock in DMSO), 3 µM Vinblastine (Sigma-Aldrich, V1377; 11 mM stock in DMSO), or 60 nM Leptomycin B (Sigma-Aldrich, L2913; 10.3 µM in 70% ethanol).

    Techniques: De-Phosphorylation Assay, Confocal Microscopy, Staining, Marker, MANN-WHITNEY

    DNA damage induces de-phosphorylation of nuclear Tau. ( a ) The effect of Etoposide and Vinblastine treatment on mouse C17.2 cells is shown by confocal microscopy upon immune staining of PFA-fixed cells with antibodies against the microtubule marker β-tubulin (in cyan) or the DNA damage marker γH2A-X (in red). ( b ) Confocal microscopic quantification of the activated kinases in the nucleus (DAPI mask). Mean percent ± sem relative to the respective controls. 2-tailed unpaired Mann-Whitney test, ****p

    Journal: Scientific Reports

    Article Title: Phosphorylation of nuclear Tau is modulated by distinct cellular pathways

    doi: 10.1038/s41598-018-36374-4

    Figure Lengend Snippet: DNA damage induces de-phosphorylation of nuclear Tau. ( a ) The effect of Etoposide and Vinblastine treatment on mouse C17.2 cells is shown by confocal microscopy upon immune staining of PFA-fixed cells with antibodies against the microtubule marker β-tubulin (in cyan) or the DNA damage marker γH2A-X (in red). ( b ) Confocal microscopic quantification of the activated kinases in the nucleus (DAPI mask). Mean percent ± sem relative to the respective controls. 2-tailed unpaired Mann-Whitney test, ****p

    Article Snippet: During the last 5 h of tetracycline incubation, C17.2 cells with inducible Tau expression were treated with 60 µM Etoposide (Abcam, ab120227; 100 mM stock in DMSO), 3 µM Vinblastine (Sigma-Aldrich, V1377; 11 mM stock in DMSO), or 60 nM Leptomycin B (Sigma-Aldrich, L2913; 10.3 µM in 70% ethanol).

    Techniques: De-Phosphorylation Assay, Confocal Microscopy, Staining, Marker, MANN-WHITNEY

    Tau depletion decreases P53 level and apoptosis. For all panels the indicated cell lines (Tau-KO are 232P cells) were treated 30 min with the indicated etoposide concentrations and recovery times. a Mean intensity ± sem of single-cell nuclear P53 staining (DAPI mask, ImageJ) is shown as fold of wt cells at basal conditions, n > 100 cells/condition distributed over five images. b Percent clCasp3-positive cells are shown as mean ± SD of five images for the untreated cells and of 15 images for etoposide-treated cells, n > 500 cells/condition. c Mean intensity ± sem of single-cell Tau staining (tubulin mask, ImageJ) for the indicated cell lines is shown as percent of mock shRNA cells (ctrl), n > 160 cells/condition distributed over five images. d Mean intensity of single-cell nuclear P53 staining quantified as in a , n > 380 cell/condition distributed over 15 images from n = 3 biological replicates. e Percent clCasp3-positive cells quantified as in b , n > 500 cell/condition. f An arbitrary threshold was applied in order to count P53-positive cells as percentage ± SD of total DAPI-positive cell number, n > 100 cells/conditions. For the comparison between the four cell lines ( a ), non-parametric independent Mann–Whitney U test for genotype (in bold) and Kruskal–Wallis pairwise comparison of treatment for cell lines with same genotype (in italics) or for each line (in vertical). For the dose-dependency ( a , b ), non-parametric independent Mann–Whitney U test between cell lines (in bold), Kruskal–Wallis pairwise comparison for each dose (in italics) and between doses (in vertical). Non-parametric independent Mann–Whitney U test ( c – e ) between control and the three Tau-KD lines (in bold) and Kruskal–Wallis pairwise comparison for each Tau-KD line (in italic) and for each treatment (in vertical). Unpaired two-tailed t test with Welch’s correction ( f ).

    Journal: Communications Biology

    Article Title: Tau affects P53 function and cell fate during the DNA damage response

    doi: 10.1038/s42003-020-0975-4

    Figure Lengend Snippet: Tau depletion decreases P53 level and apoptosis. For all panels the indicated cell lines (Tau-KO are 232P cells) were treated 30 min with the indicated etoposide concentrations and recovery times. a Mean intensity ± sem of single-cell nuclear P53 staining (DAPI mask, ImageJ) is shown as fold of wt cells at basal conditions, n > 100 cells/condition distributed over five images. b Percent clCasp3-positive cells are shown as mean ± SD of five images for the untreated cells and of 15 images for etoposide-treated cells, n > 500 cells/condition. c Mean intensity ± sem of single-cell Tau staining (tubulin mask, ImageJ) for the indicated cell lines is shown as percent of mock shRNA cells (ctrl), n > 160 cells/condition distributed over five images. d Mean intensity of single-cell nuclear P53 staining quantified as in a , n > 380 cell/condition distributed over 15 images from n = 3 biological replicates. e Percent clCasp3-positive cells quantified as in b , n > 500 cell/condition. f An arbitrary threshold was applied in order to count P53-positive cells as percentage ± SD of total DAPI-positive cell number, n > 100 cells/conditions. For the comparison between the four cell lines ( a ), non-parametric independent Mann–Whitney U test for genotype (in bold) and Kruskal–Wallis pairwise comparison of treatment for cell lines with same genotype (in italics) or for each line (in vertical). For the dose-dependency ( a , b ), non-parametric independent Mann–Whitney U test between cell lines (in bold), Kruskal–Wallis pairwise comparison for each dose (in italics) and between doses (in vertical). Non-parametric independent Mann–Whitney U test ( c – e ) between control and the three Tau-KD lines (in bold) and Kruskal–Wallis pairwise comparison for each Tau-KD line (in italic) and for each treatment (in vertical). Unpaired two-tailed t test with Welch’s correction ( f ).

    Article Snippet: Drug treatments Etoposide (100 mM stock in DMSO; ab120227, Abcam) treatment was followed by three washes with complete DMEM and cells were allowed to recover for the indicated times.

    Techniques: Staining, shRNA, MANN-WHITNEY, Two Tailed Test

    Tau does not directly interact with P53 . Cell lysates of SH-SY5Y treated with 10 μM of MG132 to stabilize P53 expression, without (ctrl) or with (eto) a 30 min pre-treatment with 60 μM etoposide, were subjected to immune precipitation of endogenous P53 with a rabbit antibody (P53) or with a rabbit GFP antibody as negative IP control (GFP). Western blot analysis for co-precipitation of MDM2 or Tau with the respective mouse antibodies as indicated. The blots on the top show the analysis of the starting material (cell lysates), those on the bottom the immunoprecipitation (IP). The P53 blots are entirely shown, whereas for MDM2 and Tau, the blots were cut between the 55 kDa and the 95 kDa protein size markers and analyzed separately.

    Journal: Communications Biology

    Article Title: Tau affects P53 function and cell fate during the DNA damage response

    doi: 10.1038/s42003-020-0975-4

    Figure Lengend Snippet: Tau does not directly interact with P53 . Cell lysates of SH-SY5Y treated with 10 μM of MG132 to stabilize P53 expression, without (ctrl) or with (eto) a 30 min pre-treatment with 60 μM etoposide, were subjected to immune precipitation of endogenous P53 with a rabbit antibody (P53) or with a rabbit GFP antibody as negative IP control (GFP). Western blot analysis for co-precipitation of MDM2 or Tau with the respective mouse antibodies as indicated. The blots on the top show the analysis of the starting material (cell lysates), those on the bottom the immunoprecipitation (IP). The P53 blots are entirely shown, whereas for MDM2 and Tau, the blots were cut between the 55 kDa and the 95 kDa protein size markers and analyzed separately.

    Article Snippet: Drug treatments Etoposide (100 mM stock in DMSO; ab120227, Abcam) treatment was followed by three washes with complete DMEM and cells were allowed to recover for the indicated times.

    Techniques: Expressing, Western Blot, Immunoprecipitation

    Differential regulation of P53 transcription targets . Extracted RNA from parental (wt) and 232P (Tau-KO) cells in a or from control shRNA plasmid (ctrl) and 3127 (Tau-KD) cells in b at basal conditions or after 30 min 60 μM etoposide and 6 h recovery, was subjected to reverse-transcription and qPCR with primers specific for the indicated transcripts. Mean ± SD of relative mRNA levels ( n = 3) shown as fold of the respective basal conditions for parental or control cells. Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (horizontal), multiple Sidak pairwise comparisons for treatment for each line (in vertical).

    Journal: Communications Biology

    Article Title: Tau affects P53 function and cell fate during the DNA damage response

    doi: 10.1038/s42003-020-0975-4

    Figure Lengend Snippet: Differential regulation of P53 transcription targets . Extracted RNA from parental (wt) and 232P (Tau-KO) cells in a or from control shRNA plasmid (ctrl) and 3127 (Tau-KD) cells in b at basal conditions or after 30 min 60 μM etoposide and 6 h recovery, was subjected to reverse-transcription and qPCR with primers specific for the indicated transcripts. Mean ± SD of relative mRNA levels ( n = 3) shown as fold of the respective basal conditions for parental or control cells. Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (horizontal), multiple Sidak pairwise comparisons for treatment for each line (in vertical).

    Article Snippet: Drug treatments Etoposide (100 mM stock in DMSO; ab120227, Abcam) treatment was followed by three washes with complete DMEM and cells were allowed to recover for the indicated times.

    Techniques: shRNA, Plasmid Preparation, Real-time Polymerase Chain Reaction

    Role of P53 and MDM2 modifications for P53 function and stability . a , b Parental (wt) or 232P (Tau-KO) cells treated 30 min without (basal) or with 60 μM etoposide and recovered for 6 h in the absence (eto) or presence of 10 µg/mL KU-55933 and/or 5 µg/mL nutlin-3. a Mean intensity ± sem of single-cell nuclear P53 or MDM2 (DAPI mask, ImageJ) shown as fold of basal conditions, n > 100 cells/condition distributed over five images. b Percent clCasp3-positive cells shown as mean ± SD of five images (basal) or 15 images (treatments), n > 500 cells/condition. Non-parametric independent samples test and Kruskal–Wallis pairwise comparison between cell lines (in bold) or for treatment for each cell line (in vertical). c Western bot analysis of P53 in parental (wt) or 232 P (Tau-KO) cells at basal conditions, after 30 min 60 μM etoposide and 4 h recovery without or with 10 μM MG132. GAPDH served as loading control. d Parental (wt) or 232P (Tau-KO) cells pre-treated for 30 min with 60 μM etoposide followed by 4 h with 10 μM MG132, were incubated with 25 μM of cycloheximide (CHX) for the indicated chase times. Single-cell nuclear P53 or nuclear MDM2 (DAPI mask, ImageJ) shown as fold of wt cells at basal conditions. Mean intensity ± sem of n > 100 cells/condition distributed over five images. Independent measures ordinary two-way ANOVA, source of variation for cell lines (bold), multiple Bonferroni pairwise comparisons of treatment for each line (in italic). e Parental (wt) or (Tau-KO) 232P cells treated for 30 min with 60 μM etoposide and 6 h recovery analyzed with a 90 kDa MDM2 rabbit antibody (green and middle panel with GAPDH as loading control) or a 60 and 90 kDa MDM2 mouse antibody (red and bottom panel).

    Journal: Communications Biology

    Article Title: Tau affects P53 function and cell fate during the DNA damage response

    doi: 10.1038/s42003-020-0975-4

    Figure Lengend Snippet: Role of P53 and MDM2 modifications for P53 function and stability . a , b Parental (wt) or 232P (Tau-KO) cells treated 30 min without (basal) or with 60 μM etoposide and recovered for 6 h in the absence (eto) or presence of 10 µg/mL KU-55933 and/or 5 µg/mL nutlin-3. a Mean intensity ± sem of single-cell nuclear P53 or MDM2 (DAPI mask, ImageJ) shown as fold of basal conditions, n > 100 cells/condition distributed over five images. b Percent clCasp3-positive cells shown as mean ± SD of five images (basal) or 15 images (treatments), n > 500 cells/condition. Non-parametric independent samples test and Kruskal–Wallis pairwise comparison between cell lines (in bold) or for treatment for each cell line (in vertical). c Western bot analysis of P53 in parental (wt) or 232 P (Tau-KO) cells at basal conditions, after 30 min 60 μM etoposide and 4 h recovery without or with 10 μM MG132. GAPDH served as loading control. d Parental (wt) or 232P (Tau-KO) cells pre-treated for 30 min with 60 μM etoposide followed by 4 h with 10 μM MG132, were incubated with 25 μM of cycloheximide (CHX) for the indicated chase times. Single-cell nuclear P53 or nuclear MDM2 (DAPI mask, ImageJ) shown as fold of wt cells at basal conditions. Mean intensity ± sem of n > 100 cells/condition distributed over five images. Independent measures ordinary two-way ANOVA, source of variation for cell lines (bold), multiple Bonferroni pairwise comparisons of treatment for each line (in italic). e Parental (wt) or (Tau-KO) 232P cells treated for 30 min with 60 μM etoposide and 6 h recovery analyzed with a 90 kDa MDM2 rabbit antibody (green and middle panel with GAPDH as loading control) or a 60 and 90 kDa MDM2 mouse antibody (red and bottom panel).

    Article Snippet: Drug treatments Etoposide (100 mM stock in DMSO; ab120227, Abcam) treatment was followed by three washes with complete DMEM and cells were allowed to recover for the indicated times.

    Techniques: Western Blot, Incubation

    Tau deficiency confers resistance to etoposide-induced apoptosis . Scheme representing the design of the experiment with parental and 231A (Tau) cells compared to 232P and 231K (Tau-KO) cells treated 30 min with 60 μM etoposide and recovered as indicated before analysis. LDH and MTS values are shown as percentage of parental cells (wt), mean ± SD of five biological replicates. To measure activation of apoptosis, percent positive cells for cleaved-caspase-3 (clCasp3) is determined on confocal microscope images and normalized for total DAPI-positive cells, mean ± SD of five images for the untreated cells (ctrl) and of 15 images for etoposide-treated cells (60 μM eto), n > 500 cells/condition, representative experiment of n > 3 biological replicates. Activated clCasp3 was also analyzed by western blot with GAPDH as loading control and 15 and 17 kDa clCasp3 quantified by normalization with GAPDH, mean ± SD ( n = 3 biological triplicates). Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (in bold), multiple Bonferroni pairwise comparisons for treatment between lines (in italics) or for each line (in vertical).

    Journal: Communications Biology

    Article Title: Tau affects P53 function and cell fate during the DNA damage response

    doi: 10.1038/s42003-020-0975-4

    Figure Lengend Snippet: Tau deficiency confers resistance to etoposide-induced apoptosis . Scheme representing the design of the experiment with parental and 231A (Tau) cells compared to 232P and 231K (Tau-KO) cells treated 30 min with 60 μM etoposide and recovered as indicated before analysis. LDH and MTS values are shown as percentage of parental cells (wt), mean ± SD of five biological replicates. To measure activation of apoptosis, percent positive cells for cleaved-caspase-3 (clCasp3) is determined on confocal microscope images and normalized for total DAPI-positive cells, mean ± SD of five images for the untreated cells (ctrl) and of 15 images for etoposide-treated cells (60 μM eto), n > 500 cells/condition, representative experiment of n > 3 biological replicates. Activated clCasp3 was also analyzed by western blot with GAPDH as loading control and 15 and 17 kDa clCasp3 quantified by normalization with GAPDH, mean ± SD ( n = 3 biological triplicates). Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (in bold), multiple Bonferroni pairwise comparisons for treatment between lines (in italics) or for each line (in vertical).

    Article Snippet: Drug treatments Etoposide (100 mM stock in DMSO; ab120227, Abcam) treatment was followed by three washes with complete DMEM and cells were allowed to recover for the indicated times.

    Techniques: Activation Assay, Microscopy, Western Blot

    Reduced P53 in Tau-KO cells is not caused by DDR activation . For all panels, parental (wt) or 232P (Tau-KO) cells were treated 30 min with the indicated etoposide concentrations and recovery times. a Mean intensity ± SD of γH2A-X staining normalized for DAPI staining by in-cell western is shown as fold of wt cells at basal conditions, n = 5 of biological replicates in a 96-well plate. Non-parametric independent Mann–Whitney U test between lines (in bold), or for each dose between lines (in italics). b Olive moment in the Comet assay is shown as mean ± sem, n = 84–146 cells/condition. c , d Mean intensity ± sem of single-cell nuclear γH2A-X, pATM or pChk2 staining (DAPI mask, ImageJ) is shown as fold of wt cells at basal conditions, n > 100 cells/condition distributed over five images. Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (in bold), multiple Bonferroni pairwise comparisons of each condition between lines (in italics) and of time points for each line ( b , d , in vertical).

    Journal: Communications Biology

    Article Title: Tau affects P53 function and cell fate during the DNA damage response

    doi: 10.1038/s42003-020-0975-4

    Figure Lengend Snippet: Reduced P53 in Tau-KO cells is not caused by DDR activation . For all panels, parental (wt) or 232P (Tau-KO) cells were treated 30 min with the indicated etoposide concentrations and recovery times. a Mean intensity ± SD of γH2A-X staining normalized for DAPI staining by in-cell western is shown as fold of wt cells at basal conditions, n = 5 of biological replicates in a 96-well plate. Non-parametric independent Mann–Whitney U test between lines (in bold), or for each dose between lines (in italics). b Olive moment in the Comet assay is shown as mean ± sem, n = 84–146 cells/condition. c , d Mean intensity ± sem of single-cell nuclear γH2A-X, pATM or pChk2 staining (DAPI mask, ImageJ) is shown as fold of wt cells at basal conditions, n > 100 cells/condition distributed over five images. Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (in bold), multiple Bonferroni pairwise comparisons of each condition between lines (in italics) and of time points for each line ( b , d , in vertical).

    Article Snippet: Drug treatments Etoposide (100 mM stock in DMSO; ab120227, Abcam) treatment was followed by three washes with complete DMEM and cells were allowed to recover for the indicated times.

    Techniques: Activation Assay, Staining, In-Cell ELISA, MANN-WHITNEY, Single Cell Gel Electrophoresis

    Tau depletion increases cellular senescence . a Scheme of the procedure followed to assess cellular senescence upon 30 min treatment with 60 μM etoposide followed by 3 days of recovery. b Quantification of p21 amount in cell lysates by western blot in parental (wt) or 232P (Tau-KO) cells under control conditions (ctrl) or following etoposide treatment (60 µM eto) normalized for GAPDH, mean ± SD of three biological replicates. Quantification of mean cell area and percent positive cells for senescence-associated β-galactosidase (SA- βGal) determined with a high-content microscope scanner, mean ± sem of four (Tau-KO cells) or three (Tau-KD) independent experiments, n > 8000 cells. Data are shown as fold of wt cells at basal conditions. c Same as in b for mock shRNA (ctrl) or Tau 3127 shRNA (Tau-KD) cells. d Representative images of SA-βGal staining (in blue), bright-field, scale bar = 100 μm. Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (in bold), multiple Bonferroni pairwise comparisons for treatment between lines (in italics) or for each line (in vertical).

    Journal: Communications Biology

    Article Title: Tau affects P53 function and cell fate during the DNA damage response

    doi: 10.1038/s42003-020-0975-4

    Figure Lengend Snippet: Tau depletion increases cellular senescence . a Scheme of the procedure followed to assess cellular senescence upon 30 min treatment with 60 μM etoposide followed by 3 days of recovery. b Quantification of p21 amount in cell lysates by western blot in parental (wt) or 232P (Tau-KO) cells under control conditions (ctrl) or following etoposide treatment (60 µM eto) normalized for GAPDH, mean ± SD of three biological replicates. Quantification of mean cell area and percent positive cells for senescence-associated β-galactosidase (SA- βGal) determined with a high-content microscope scanner, mean ± sem of four (Tau-KO cells) or three (Tau-KD) independent experiments, n > 8000 cells. Data are shown as fold of wt cells at basal conditions. c Same as in b for mock shRNA (ctrl) or Tau 3127 shRNA (Tau-KD) cells. d Representative images of SA-βGal staining (in blue), bright-field, scale bar = 100 μm. Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (in bold), multiple Bonferroni pairwise comparisons for treatment between lines (in italics) or for each line (in vertical).

    Article Snippet: Drug treatments Etoposide (100 mM stock in DMSO; ab120227, Abcam) treatment was followed by three washes with complete DMEM and cells were allowed to recover for the indicated times.

    Techniques: Western Blot, Microscopy, shRNA, Staining

    OTIs designed against a patient-observed PML-RARA translocation increase DNA cleavage mediated by human type II topoisomerases. ( A ) Sequences of the top and bottom strands of each PML-RARA duplex are shown. The blue portion corresponds to the segment derived from the PML gene, and the orange portion corresponds to the segment derived from the RARA gene. The yellow box indicates the position of the tethered etoposide core on each OTI (bottom strand). The OTIs were 50, 30 or 20 bases in length (black lines below the diagram). Arrows indicate sites of DNA cleavage induced by free etoposide (blue) and the translocation OTIs (yellow). ( B ) Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα, left) and topoisomerase IIβ (hTIIβ, right) of the radiolabeled, unmodified PML-RARA top/target strand hybridized to an unmodified PML-RARA bottom strand in the presence of free etoposide or of the radiolabeled PML-RARA top strand hybridized to a 50-mer, 30-mer or 20-mer PML-RARA OTI bottom strand. Lanes 1–5 contain the unmodified PML-RARA duplex in the absence of enzyme, or in the presence of enzyme and 0–500 μM free etoposide. Lanes 7 and 8 contain the unmodified PML-RARA top strand hybridized with the 50-mer OTI. Lanes 10 and 11 contain the unmodified top strand hybridized with the 30-mer OTI. Lanes 13 and 14 contain the unmodified top strand hybridized with the 20-mer OTI. Lanes 6, 10, 13, and 15 contain reference (R) oligonucleotides 24, 23, 20 and 19 bases in length. Gels are representative of at least three independent experiments. ( C ) Quantification of the relative levels of enzyme-mediated DNA cleavage. DNA cleavage at each site is normalized to the cleavage observed at site 24–25 in reactions containing an unmodified duplex in the absence of etoposide (lane 2). Results with unmodified PML – RARA duplex in the presence of 500 μM free etoposide (blue) or with unmodified top strand hybridized with 50-mer OTI (yellow), 30-mer OTI (orange), or 20-mer OTI (green) bottom strand are shown. Error bars represent the standard deviation of at least three independent experiments. Significance was determined by paired t -tests. P -values are indicated by asterisks (* P

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: OTIs designed against a patient-observed PML-RARA translocation increase DNA cleavage mediated by human type II topoisomerases. ( A ) Sequences of the top and bottom strands of each PML-RARA duplex are shown. The blue portion corresponds to the segment derived from the PML gene, and the orange portion corresponds to the segment derived from the RARA gene. The yellow box indicates the position of the tethered etoposide core on each OTI (bottom strand). The OTIs were 50, 30 or 20 bases in length (black lines below the diagram). Arrows indicate sites of DNA cleavage induced by free etoposide (blue) and the translocation OTIs (yellow). ( B ) Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα, left) and topoisomerase IIβ (hTIIβ, right) of the radiolabeled, unmodified PML-RARA top/target strand hybridized to an unmodified PML-RARA bottom strand in the presence of free etoposide or of the radiolabeled PML-RARA top strand hybridized to a 50-mer, 30-mer or 20-mer PML-RARA OTI bottom strand. Lanes 1–5 contain the unmodified PML-RARA duplex in the absence of enzyme, or in the presence of enzyme and 0–500 μM free etoposide. Lanes 7 and 8 contain the unmodified PML-RARA top strand hybridized with the 50-mer OTI. Lanes 10 and 11 contain the unmodified top strand hybridized with the 30-mer OTI. Lanes 13 and 14 contain the unmodified top strand hybridized with the 20-mer OTI. Lanes 6, 10, 13, and 15 contain reference (R) oligonucleotides 24, 23, 20 and 19 bases in length. Gels are representative of at least three independent experiments. ( C ) Quantification of the relative levels of enzyme-mediated DNA cleavage. DNA cleavage at each site is normalized to the cleavage observed at site 24–25 in reactions containing an unmodified duplex in the absence of etoposide (lane 2). Results with unmodified PML – RARA duplex in the presence of 500 μM free etoposide (blue) or with unmodified top strand hybridized with 50-mer OTI (yellow), 30-mer OTI (orange), or 20-mer OTI (green) bottom strand are shown. Error bars represent the standard deviation of at least three independent experiments. Significance was determined by paired t -tests. P -values are indicated by asterisks (* P

    Article Snippet: Synthesis of the activated etoposide core, shown in the top portion of Figure , started with commercially available DEPT ( 1 ) (ABCAM Biochemicals).

    Techniques: Translocation Assay, Derivative Assay, Standard Deviation

    Structure-guided design of an OTI. ( A ) Schematic illustrating domains of type II topoisomerases used to determine the crystal structure of human topoisomerase IIβ covalently attached to DNA (green) in the presence of etoposide (orange). Domains pictured are TOPRIM (Top), winged helix domain (WHD), tower domain (Tow), and exit gate domain (Ex). ( B ) Schematic of topoisomerase II function. Protein protomer subunits are shown in blue and gray. T DNA, transport double helix (black); G DNA, gate double helix (green). ( C, D ) Detail from the crystal structure of a topoisomerase IIβ cleavage complex with two bound etoposide molecules (orange) stabilizing a double-stranded DNA (green) break; PDB code 3QX3. For clarity, in panel C, only the Cα trace of the protein subunits (blue and black lines) and catalytic tyrosines (blue and gray sticks) are shown. In panel D, only the catalytic tyrosine residues that cleave the DNA are shown. The conventional numbering scheme used for DNA cleavage complexes formed by type II topoisomerases is shown. The enzyme cleaves between the -1 and the +1 on each strand. The numbering on the two strands in the double helix is differentiated by the presence or absence of asterisks. The catalytic tyrosine residues are covalently attached to the DNA at the +1 positions. ( E , F ) Model of a cleavage complex with one bound etoposide molecule stabilizing a single-stranded DNA break. The cleaved DNA strand is indicated by asterisks. The protein subunits shown are the same as those in C and D. ( G ) Chemical (left) and modeled (right) structure of the etoposide core (DEPT) linked to the pyrimidine base. ( H ) OTI28 (orange strand) modeled with the modified cytosine base in the +5* position, stabilizing DNA scission at the 23–24 site (–1 to +1) on the cleaved target strand (green). Structural figures were drawn with Pymol (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC).

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: Structure-guided design of an OTI. ( A ) Schematic illustrating domains of type II topoisomerases used to determine the crystal structure of human topoisomerase IIβ covalently attached to DNA (green) in the presence of etoposide (orange). Domains pictured are TOPRIM (Top), winged helix domain (WHD), tower domain (Tow), and exit gate domain (Ex). ( B ) Schematic of topoisomerase II function. Protein protomer subunits are shown in blue and gray. T DNA, transport double helix (black); G DNA, gate double helix (green). ( C, D ) Detail from the crystal structure of a topoisomerase IIβ cleavage complex with two bound etoposide molecules (orange) stabilizing a double-stranded DNA (green) break; PDB code 3QX3. For clarity, in panel C, only the Cα trace of the protein subunits (blue and black lines) and catalytic tyrosines (blue and gray sticks) are shown. In panel D, only the catalytic tyrosine residues that cleave the DNA are shown. The conventional numbering scheme used for DNA cleavage complexes formed by type II topoisomerases is shown. The enzyme cleaves between the -1 and the +1 on each strand. The numbering on the two strands in the double helix is differentiated by the presence or absence of asterisks. The catalytic tyrosine residues are covalently attached to the DNA at the +1 positions. ( E , F ) Model of a cleavage complex with one bound etoposide molecule stabilizing a single-stranded DNA break. The cleaved DNA strand is indicated by asterisks. The protein subunits shown are the same as those in C and D. ( G ) Chemical (left) and modeled (right) structure of the etoposide core (DEPT) linked to the pyrimidine base. ( H ) OTI28 (orange strand) modeled with the modified cytosine base in the +5* position, stabilizing DNA scission at the 23–24 site (–1 to +1) on the cleaved target strand (green). Structural figures were drawn with Pymol (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC).

    Article Snippet: Synthesis of the activated etoposide core, shown in the top portion of Figure , started with commercially available DEPT ( 1 ) (ABCAM Biochemicals).

    Techniques: Modification

    ). ( A ) Cleavage between bases 24–25 is depicted on the target (top) strand (green). ( B ) Cleavage between bases 23–24 is depicted on the target strand (green). The bottom (OTI) strand is shown in orange. The tethered etoposide core is shown in yellow (carbons, yellow; nitrogen, blue; oxygen, red). A Cα trace is shown for the two topoisomerase II subunits (blue and black lines) in the top panels. The bottom panels include a semi-transparent molecular surface, illustrating that the linker does not clash with the protein. The sequence diagram (middle) shows the position of the tethered etoposide core on OTI28 (yellow box). Black arrows indicate the cleavage sites.

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: ). ( A ) Cleavage between bases 24–25 is depicted on the target (top) strand (green). ( B ) Cleavage between bases 23–24 is depicted on the target strand (green). The bottom (OTI) strand is shown in orange. The tethered etoposide core is shown in yellow (carbons, yellow; nitrogen, blue; oxygen, red). A Cα trace is shown for the two topoisomerase II subunits (blue and black lines) in the top panels. The bottom panels include a semi-transparent molecular surface, illustrating that the linker does not clash with the protein. The sequence diagram (middle) shows the position of the tethered etoposide core on OTI28 (yellow box). Black arrows indicate the cleavage sites.

    Article Snippet: Synthesis of the activated etoposide core, shown in the top portion of Figure , started with commercially available DEPT ( 1 ) (ABCAM Biochemicals).

    Techniques: Sequencing

    OTI28 inhibits DNA ligation and stabilizes cleavage complexes similarly to free etoposide. ( A ) Enzyme-mediated ligation of DNA. ( B ) Persistence of cleavage complexes. For both A and B, cleavage results of the unmodified PML duplex in the presence of 500 μM free etoposide are shown in blue and those with an unmodified PML top/target strand hybridized to OTI28 are shown in yellow. Error bars represent the standard deviation of at least three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: OTI28 inhibits DNA ligation and stabilizes cleavage complexes similarly to free etoposide. ( A ) Enzyme-mediated ligation of DNA. ( B ) Persistence of cleavage complexes. For both A and B, cleavage results of the unmodified PML duplex in the presence of 500 μM free etoposide are shown in blue and those with an unmodified PML top/target strand hybridized to OTI28 are shown in yellow. Error bars represent the standard deviation of at least three independent experiments.

    Article Snippet: Synthesis of the activated etoposide core, shown in the top portion of Figure , started with commercially available DEPT ( 1 ) (ABCAM Biochemicals).

    Techniques: DNA Ligation, Ligation, Standard Deviation

    An oligonucleotide-linked etoposide core increases topoisomerase II-mediated DNA cleavage. ( A ) The central 30 base pairs of a double stranded 50-mer oligonucleotide sequence corresponding to bases 1471–1500 (top strand) of PML intron 6 is shown. The yellow box denotes the position of the tethered etoposide core and linker moieties on OTI28 or LIN28. Arrows indicate sites of DNA cleavage induced by free etoposide (blue) or OTI28 (yellow). ( B ) Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα, left) and topoisomerase IIβ (hTIIβ, right) of the radiolabeled, unmodified PML top strand hybridized to an unmodified PML bottom strand in the presence of free etoposide or hybridized to OTI28 (bottom strand). For each gel, lane 1 contains the unmodified PML oligonucleotide. Lanes 2–5 contain the unmodified PML duplex treated with 0–500 μM free etoposide. Lanes 7 and 8 contain the unmodified PML top strand hybridized with OTI28. Lanes 10 and 11 contain the unmodified top strand duplexed with LIN28 (bottom strand oligonucleotide that contains the linker at position 28 with no attached etoposide core). Lanes 6, 9, and 12 contain reference (R) oligonucleotides that were 24, 23 and 19 bases in length. Gels are representative of at least three independent experiments. ( C ) Quantification of the relative levels of DNA cleavage mediated by topoisomerase IIα (left) and topoisomerase IIβ (right). DNA cleavage at each site was normalized to the cleavage observed at site 24–25 in reactions containing unmodified duplex in the absence of etoposide (lane 2). Cleavage results of the unmodified duplex in the presence of 500 μM free etoposide are shown in blue (lane 5) and those with an unmodified top strand hybridized to OTI28 are shown in yellow (lane 8). Error bars represent the standard error of the mean of an average of two to five independent experiments. Significance was determined by paired t-tests. P -values are indicated by asterisks (* P

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: An oligonucleotide-linked etoposide core increases topoisomerase II-mediated DNA cleavage. ( A ) The central 30 base pairs of a double stranded 50-mer oligonucleotide sequence corresponding to bases 1471–1500 (top strand) of PML intron 6 is shown. The yellow box denotes the position of the tethered etoposide core and linker moieties on OTI28 or LIN28. Arrows indicate sites of DNA cleavage induced by free etoposide (blue) or OTI28 (yellow). ( B ) Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα, left) and topoisomerase IIβ (hTIIβ, right) of the radiolabeled, unmodified PML top strand hybridized to an unmodified PML bottom strand in the presence of free etoposide or hybridized to OTI28 (bottom strand). For each gel, lane 1 contains the unmodified PML oligonucleotide. Lanes 2–5 contain the unmodified PML duplex treated with 0–500 μM free etoposide. Lanes 7 and 8 contain the unmodified PML top strand hybridized with OTI28. Lanes 10 and 11 contain the unmodified top strand duplexed with LIN28 (bottom strand oligonucleotide that contains the linker at position 28 with no attached etoposide core). Lanes 6, 9, and 12 contain reference (R) oligonucleotides that were 24, 23 and 19 bases in length. Gels are representative of at least three independent experiments. ( C ) Quantification of the relative levels of DNA cleavage mediated by topoisomerase IIα (left) and topoisomerase IIβ (right). DNA cleavage at each site was normalized to the cleavage observed at site 24–25 in reactions containing unmodified duplex in the absence of etoposide (lane 2). Cleavage results of the unmodified duplex in the presence of 500 μM free etoposide are shown in blue (lane 5) and those with an unmodified top strand hybridized to OTI28 are shown in yellow (lane 8). Error bars represent the standard error of the mean of an average of two to five independent experiments. Significance was determined by paired t-tests. P -values are indicated by asterisks (* P

    Article Snippet: Synthesis of the activated etoposide core, shown in the top portion of Figure , started with commercially available DEPT ( 1 ) (ABCAM Biochemicals).

    Techniques: Sequencing

    OTI28 induces lower levels of DNA cleavage mediated by an etoposide-resistant mutant yeast topoisomerase II (H1011Y) as compared to wild-type yeast topoisomerase II. Quantification of the relative levels of enzyme-mediated DNA cleavage at site 24–25 (indicated as the band labeled 24 in the inset) mediated by wild-type (yTop2WT) and H1011Y mutant (yTop2H1011Y) yeast topoisomerase II on an unmodified PML top strand hybridized to OTI28 (graph: +enz, red; inset: +WT, +H1011Y). DNA cleavage is normalized to background levels of DNA when no enzyme is present (graph: -enz, blue; inset: -WT, -H1011Y). Error bars represent the standard deviation of three independent experiments. Significance was determined by a paired t -test. P -values are indicated by asterisks (*** P

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: OTI28 induces lower levels of DNA cleavage mediated by an etoposide-resistant mutant yeast topoisomerase II (H1011Y) as compared to wild-type yeast topoisomerase II. Quantification of the relative levels of enzyme-mediated DNA cleavage at site 24–25 (indicated as the band labeled 24 in the inset) mediated by wild-type (yTop2WT) and H1011Y mutant (yTop2H1011Y) yeast topoisomerase II on an unmodified PML top strand hybridized to OTI28 (graph: +enz, red; inset: +WT, +H1011Y). DNA cleavage is normalized to background levels of DNA when no enzyme is present (graph: -enz, blue; inset: -WT, -H1011Y). Error bars represent the standard deviation of three independent experiments. Significance was determined by a paired t -test. P -values are indicated by asterisks (*** P

    Article Snippet: Synthesis of the activated etoposide core, shown in the top portion of Figure , started with commercially available DEPT ( 1 ) (ABCAM Biochemicals).

    Techniques: Mutagenesis, Labeling, Standard Deviation

    Moving the position of the linked etoposide core along the bottom strand (OTI) sequence alters the topoisomerase II-mediated cleavage pattern of the top strand. ( A ) Sequences of the top/target and bottom PML strands are shown. The different-colored boxes indicate the position of the tethered etoposide core in each OTI (bottom strand), including OTI28 (yellow), OTI29 (orange), OTI33 (green), and OTI23 (purple). Arrows indicate the cleavage sites induced by each OTI (shown by corresponding colors). Large arrows indicate the major site of cleavage. T and B (first column) indicate top and bottom strands, respectively. ( B ) Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα, left) and topoisomerase IIβ (hTIIβ, right) of the radiolabeled, unmodified PML top strand hybridized to an unmodified PML bottom strand in the presence of 0–500 μM free etoposide (lanes 2–3) or hybridized to the bottom strands OTI28 (lanes 5–6), OTI29 (lanes 8–9), OTI33 (lanes 11–12), or OTI23 (lanes 14–15). For each gel, lane 1 contains an unmodified PML duplex. Reference (R) oligonucleotides are 24, 23, 20 and 19 bases long. Gels are representative of at least three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: Moving the position of the linked etoposide core along the bottom strand (OTI) sequence alters the topoisomerase II-mediated cleavage pattern of the top strand. ( A ) Sequences of the top/target and bottom PML strands are shown. The different-colored boxes indicate the position of the tethered etoposide core in each OTI (bottom strand), including OTI28 (yellow), OTI29 (orange), OTI33 (green), and OTI23 (purple). Arrows indicate the cleavage sites induced by each OTI (shown by corresponding colors). Large arrows indicate the major site of cleavage. T and B (first column) indicate top and bottom strands, respectively. ( B ) Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα, left) and topoisomerase IIβ (hTIIβ, right) of the radiolabeled, unmodified PML top strand hybridized to an unmodified PML bottom strand in the presence of 0–500 μM free etoposide (lanes 2–3) or hybridized to the bottom strands OTI28 (lanes 5–6), OTI29 (lanes 8–9), OTI33 (lanes 11–12), or OTI23 (lanes 14–15). For each gel, lane 1 contains an unmodified PML duplex. Reference (R) oligonucleotides are 24, 23, 20 and 19 bases long. Gels are representative of at least three independent experiments.

    Article Snippet: Synthesis of the activated etoposide core, shown in the top portion of Figure , started with commercially available DEPT ( 1 ) (ABCAM Biochemicals).

    Techniques: Sequencing

    An oligonucleotide with an abasic site analog at position 28 generates a different DNA cleavage pattern than does OTI28. Lanes 1–3 contain a radiolabeled unmodified PML top/target strand hybridized to an unmodified PML bottom strand in the absence of enzyme, or in the presence of enzyme and 0–500 μM free etoposide. Lanes 4 and 5 contain a radiolabeled PML top strand hybridized with OTI28 (bottom strand). Lanes 6 and 7 contain a radiolabeled unmodified PML top strand hybridized to a bottom strand oligonucleotide containing an abasic site analog at position 28 (AP28). Lane 8 contains reference (R) oligonucleotides that are 24, 23 and 19 bases in length. The gel is representative of at least three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: An oligonucleotide with an abasic site analog at position 28 generates a different DNA cleavage pattern than does OTI28. Lanes 1–3 contain a radiolabeled unmodified PML top/target strand hybridized to an unmodified PML bottom strand in the absence of enzyme, or in the presence of enzyme and 0–500 μM free etoposide. Lanes 4 and 5 contain a radiolabeled PML top strand hybridized with OTI28 (bottom strand). Lanes 6 and 7 contain a radiolabeled unmodified PML top strand hybridized to a bottom strand oligonucleotide containing an abasic site analog at position 28 (AP28). Lane 8 contains reference (R) oligonucleotides that are 24, 23 and 19 bases in length. The gel is representative of at least three independent experiments.

    Article Snippet: Synthesis of the activated etoposide core, shown in the top portion of Figure , started with commercially available DEPT ( 1 ) (ABCAM Biochemicals).

    Techniques:

    OTIs that incorporate an APL patient-derived PML-RARA translocation sequence do not increase DNA cleavage mediated by human type II topoisomerases when they are hybridized with the parental PML or RARA sequences. Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα) ( A ) and topoisomerase IIβ (hTIIβ) ( B ) of the radiolabeled top strand of an unmodified PML-RARA duplex in the presence of free etoposide, a radiolabeled, unmodified PML or RARA top strand hybridized to a PML-RARA bottom strand in the presence of free etoposide, or a radiolabeled, unmodified PML or RARA top strand hybridized to a 50-mer OTI, a 30-mer OTI, or a 20-mer PML-RARA OTI bottom strand. For each gel, lanes 1–3 contain unmodified PML-RARA top/target strand hybridized to the unmodified 50-mer PML-RARA bottom strand, in the absence of enzyme, or in the presence of enzyme and 0–500 μM etoposide. Lanes 4–6 contain unmodified parental PML top/target strand hybridized to the unmodified 50-mer PML-RARA bottom strand, in the absence of enzyme, or in the presence of enzyme and 0–500 μM etoposide. Lanes 7–12 contain the unmodified parental PML top strand hybridized to the 50-mer, 30-mer or 20-mer PML-RARA OTI bottom strand in the absence or presence of enzyme. Lanes 14–16 contain unmodified parental RARA top strand hybridized to the 50-mer PML-RARA bottom strand, in the absence of enzyme, or in the presence of enzyme and 0–500 μM etoposide. Lanes 17–22 contain the unmodified parental RARA top strand hybridized to the 50-mer, 30-mer, or 20-mer PML-RARA OTI bottom strand in the absence or presence of enzyme. Lanes 13 and 23 contain a combination of reference (R) oligonucleotides 24, 23, 20 and 19 bases in length. Gels are representative of at least three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: OTIs that incorporate an APL patient-derived PML-RARA translocation sequence do not increase DNA cleavage mediated by human type II topoisomerases when they are hybridized with the parental PML or RARA sequences. Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα) ( A ) and topoisomerase IIβ (hTIIβ) ( B ) of the radiolabeled top strand of an unmodified PML-RARA duplex in the presence of free etoposide, a radiolabeled, unmodified PML or RARA top strand hybridized to a PML-RARA bottom strand in the presence of free etoposide, or a radiolabeled, unmodified PML or RARA top strand hybridized to a 50-mer OTI, a 30-mer OTI, or a 20-mer PML-RARA OTI bottom strand. For each gel, lanes 1–3 contain unmodified PML-RARA top/target strand hybridized to the unmodified 50-mer PML-RARA bottom strand, in the absence of enzyme, or in the presence of enzyme and 0–500 μM etoposide. Lanes 4–6 contain unmodified parental PML top/target strand hybridized to the unmodified 50-mer PML-RARA bottom strand, in the absence of enzyme, or in the presence of enzyme and 0–500 μM etoposide. Lanes 7–12 contain the unmodified parental PML top strand hybridized to the 50-mer, 30-mer or 20-mer PML-RARA OTI bottom strand in the absence or presence of enzyme. Lanes 14–16 contain unmodified parental RARA top strand hybridized to the 50-mer PML-RARA bottom strand, in the absence of enzyme, or in the presence of enzyme and 0–500 μM etoposide. Lanes 17–22 contain the unmodified parental RARA top strand hybridized to the 50-mer, 30-mer, or 20-mer PML-RARA OTI bottom strand in the absence or presence of enzyme. Lanes 13 and 23 contain a combination of reference (R) oligonucleotides 24, 23, 20 and 19 bases in length. Gels are representative of at least three independent experiments.

    Article Snippet: Synthesis of the activated etoposide core, shown in the top portion of Figure , started with commercially available DEPT ( 1 ) (ABCAM Biochemicals).

    Techniques: Derivative Assay, Translocation Assay, Sequencing