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  • 99
    New England Biolabs klenow dna polymerase
    Klenow Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dna fragments
    (A-B) Schematics illustrating the procedure to generate the slo <t>E366G</t> and slo loxP alleles via ends-out homologous recombination (A) and method for molecular validation (B). The slo locus is shown. Grey boxes represent 5’ and 3’ untranslated regions, black boxes represent both constitutive and alternatively spliced coding exons. Region encompassed by targeting arms is denoted by the grey dashed box. Linearized recombinogenic arms (Arm 1 and Arm 2, separated by loxP and mini- white + sequences) were liberated from a genomic p[w25 . 2] insertion as described in Supplemental Ref. 2. Potential recombinants were initially identified by the presence of non-white eye colour due to the mini- white + marker, then validated through PCR via the strategy shown in (B). Validated recombinants are denoted by a PCR product of approximately 3.3 kb amplified from single-fly genomic <t>DNA</t> that was absent in non-recombinant control DNA (-ve control).
    Dna Fragments, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25274 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dna fragments
    Performance of various hgRNAs. ( A ) The sequence of seven different hgRNAs. Transcription start site (TSS), spacer sequence, and PAM are marked with grey, blue, and pink boxes, respectively. ( B,C,D ) Cas9 is induced in cell lines with the hgRNAs from panel A integrated genomically. <t>DNA</t> samples harvested before (t=0 days) and at various points after induction are characterized by high-throughput sequencing to quantify mutations, functionality, and generated diversity for each hgRNA. See also Supplementary table 1 . ( E ) Design of four longer variants of <t>hgRNA-A21.</t> Stuffer sequences of 5, 30, 55, or 80 base pairs were added upstream of a spacer very similar to the A21 spacer to obtain the four increasingly lengthy A’ variants. ( F,G,H ) Cas9 is induced in cell lines with the hgRNAs from panel E integrated genomically. DNA samples harvested before (t=0 days) and at various points after induction are characterized by high-throughput sequencing to quantify mutations and functionality of hgRNA loci. Data points represent duplicate experiments.
    Dna Fragments, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6740 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Transgenomic wave dna fragment analysis system
    Mutation detection in the T4_promoter fragment. Three polymorphisms were detected by <t>dHPLC</t> in the T4_promoter fragment and were verified by <t>DNA</t> sequencing. The four dHPLC graphs correspond to the wild-type sequence (A), SNPs T-441C (B), SNP A-271G (C) and SNP G-259C (D). The wild-type pattern is visible in all dHPLC graphs as a 1 peak trace. SNP T-441C is identified by an unequal height 2 peak trace (B), SNP A-271G is identified by an equal height 2 peak trace (C) and SNP G-259C is identified by a 3 peak trace (D). The positions of the sequence variants in the T4_promoter fragment identified by dHPLC were determined by DNA sequencing. All SNPs were present in the heterozygous state.
    Wave Dna Fragment Analysis System, supplied by Transgenomic, used in various techniques. Bioz Stars score: 90/100, based on 577 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc dna fragments
    Notch1 and Notch3 are direct downstream targets of <t>Dlx5</t> ( A ) ChIP-seq analysis revealed Dlx5 binding sequences in Notch3 (MACS p value: −10*LOG10 = 70.54) and Irs2 (MACS p value: −10*LOG10 = 76.57) loci as depicted in screenshots from UCSC Genome Browser. Arrows point to binding intervals in each locus. ( B ) Multiple Em for Motif Elicitation (MEME) shows that the majority of the Dlx5 binding consensus site belongs to the classic binding motif (TAATT) of the Dlx family. ( C ) Irs2 promoters from human, monkey, rat and mouse contain Dlx5 binding consensus. ( D ) results of ChIP-qPCR assay confirmed that Dlx5 binds to two enhancer elements at end of Notch1 gene. ( E ) ChIP-qPCR verified that Dlx5 binds to enhancer element located in Notch3 intron 2. ( F ) schematic diagram of Notch1 and Notch3 promoter/enhancer reporter plasmids. Luciferase assay confirmed that Dlx5, but not Dlx5 mutant lacking the <t>DNA</t> binding domain, activates Notch1 ( G ) and Notch3 ( H ) promoters upon binding to their respective enhancer elements.
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    TaKaRa dna extraction kit
    Notch1 and Notch3 are direct downstream targets of <t>Dlx5</t> ( A ) ChIP-seq analysis revealed Dlx5 binding sequences in Notch3 (MACS p value: −10*LOG10 = 70.54) and Irs2 (MACS p value: −10*LOG10 = 76.57) loci as depicted in screenshots from UCSC Genome Browser. Arrows point to binding intervals in each locus. ( B ) Multiple Em for Motif Elicitation (MEME) shows that the majority of the Dlx5 binding consensus site belongs to the classic binding motif (TAATT) of the Dlx family. ( C ) Irs2 promoters from human, monkey, rat and mouse contain Dlx5 binding consensus. ( D ) results of ChIP-qPCR assay confirmed that Dlx5 binds to two enhancer elements at end of Notch1 gene. ( E ) ChIP-qPCR verified that Dlx5 binds to enhancer element located in Notch3 intron 2. ( F ) schematic diagram of Notch1 and Notch3 promoter/enhancer reporter plasmids. Luciferase assay confirmed that Dlx5, but not Dlx5 mutant lacking the <t>DNA</t> binding domain, activates Notch1 ( G ) and Notch3 ( H ) promoters upon binding to their respective enhancer elements.
    Dna Extraction Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs bst dna polymerase large fragment
    Notch1 and Notch3 are direct downstream targets of <t>Dlx5</t> ( A ) ChIP-seq analysis revealed Dlx5 binding sequences in Notch3 (MACS p value: −10*LOG10 = 70.54) and Irs2 (MACS p value: −10*LOG10 = 76.57) loci as depicted in screenshots from UCSC Genome Browser. Arrows point to binding intervals in each locus. ( B ) Multiple Em for Motif Elicitation (MEME) shows that the majority of the Dlx5 binding consensus site belongs to the classic binding motif (TAATT) of the Dlx family. ( C ) Irs2 promoters from human, monkey, rat and mouse contain Dlx5 binding consensus. ( D ) results of ChIP-qPCR assay confirmed that Dlx5 binds to two enhancer elements at end of Notch1 gene. ( E ) ChIP-qPCR verified that Dlx5 binds to enhancer element located in Notch3 intron 2. ( F ) schematic diagram of Notch1 and Notch3 promoter/enhancer reporter plasmids. Luciferase assay confirmed that Dlx5, but not Dlx5 mutant lacking the <t>DNA</t> binding domain, activates Notch1 ( G ) and Notch3 ( H ) promoters upon binding to their respective enhancer elements.
    Bst Dna Polymerase Large Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 330 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson dna fragmentation
    Altered timing affects the potency of NVP-BEZ235/doxorubicin combination therapy in SHEP NB cells. Three different treatment combinations were tested on SHEP NB cells, giving NVP-BEZ235 12 hrs prior to doxorubicin (Pre), giving both substances concurrently (Co), or giving NVP-BEZ235 12 hrs after the chemotherapeutic (Post). Importantly, the maximal incubation time with doxorubicin was kept constant at 24 hrs (earlier time points also shown in C and D). A SHEP NB cells were treated with NVP-BEZ235 and indicated concentrations of doxorubicin for 24 hrs, according to the scheme outlined above. <t>Apoptosis</t> was determined by FACS analysis of the <t>DNA</t> fragmentation of propidium iodide-stained nuclei, and percentage of specific DNA fragmentation is shown. B An alternative depiction of the data presented in A, highlighting the difference between the three NVP-BEZ235/doxorubicin combinations. For all following experiments 0.2 µg/ml doxorubicin was used. C Cells were either left untreated or treated as indicated and mitochondrial release of immunofluorescent-labeled cytochrome c was determined by FACS analysis. D Cells were either left untreated or treated as indicated. A Western blot analysis of caspase-3 processing served as surrogate read-out of caspase activation (appearance of the ∼12 kD cleavage fragment), β-actin was used as loading control. In A and B mean+s.e.m. values of three independent experiments carried out in triplicate, in C mean+s.d. of three independent experiments are shown, while in D a representative result of three independent experiments is depicted. Statistical analysis was carried out by two-sided Student's t -test; * P-value
    Dna Fragmentation, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 562 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs hifi dna assembly
    Altered timing affects the potency of NVP-BEZ235/doxorubicin combination therapy in SHEP NB cells. Three different treatment combinations were tested on SHEP NB cells, giving NVP-BEZ235 12 hrs prior to doxorubicin (Pre), giving both substances concurrently (Co), or giving NVP-BEZ235 12 hrs after the chemotherapeutic (Post). Importantly, the maximal incubation time with doxorubicin was kept constant at 24 hrs (earlier time points also shown in C and D). A SHEP NB cells were treated with NVP-BEZ235 and indicated concentrations of doxorubicin for 24 hrs, according to the scheme outlined above. <t>Apoptosis</t> was determined by FACS analysis of the <t>DNA</t> fragmentation of propidium iodide-stained nuclei, and percentage of specific DNA fragmentation is shown. B An alternative depiction of the data presented in A, highlighting the difference between the three NVP-BEZ235/doxorubicin combinations. For all following experiments 0.2 µg/ml doxorubicin was used. C Cells were either left untreated or treated as indicated and mitochondrial release of immunofluorescent-labeled cytochrome c was determined by FACS analysis. D Cells were either left untreated or treated as indicated. A Western blot analysis of caspase-3 processing served as surrogate read-out of caspase activation (appearance of the ∼12 kD cleavage fragment), β-actin was used as loading control. In A and B mean+s.e.m. values of three independent experiments carried out in triplicate, in C mean+s.d. of three independent experiments are shown, while in D a representative result of three independent experiments is depicted. Statistical analysis was carried out by two-sided Student's t -test; * P-value
    Hifi Dna Assembly, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 522 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore fluorescein fragel dna fragmentation detection kit
    Altered timing affects the potency of NVP-BEZ235/doxorubicin combination therapy in SHEP NB cells. Three different treatment combinations were tested on SHEP NB cells, giving NVP-BEZ235 12 hrs prior to doxorubicin (Pre), giving both substances concurrently (Co), or giving NVP-BEZ235 12 hrs after the chemotherapeutic (Post). Importantly, the maximal incubation time with doxorubicin was kept constant at 24 hrs (earlier time points also shown in C and D). A SHEP NB cells were treated with NVP-BEZ235 and indicated concentrations of doxorubicin for 24 hrs, according to the scheme outlined above. <t>Apoptosis</t> was determined by FACS analysis of the <t>DNA</t> fragmentation of propidium iodide-stained nuclei, and percentage of specific DNA fragmentation is shown. B An alternative depiction of the data presented in A, highlighting the difference between the three NVP-BEZ235/doxorubicin combinations. For all following experiments 0.2 µg/ml doxorubicin was used. C Cells were either left untreated or treated as indicated and mitochondrial release of immunofluorescent-labeled cytochrome c was determined by FACS analysis. D Cells were either left untreated or treated as indicated. A Western blot analysis of caspase-3 processing served as surrogate read-out of caspase activation (appearance of the ∼12 kD cleavage fragment), β-actin was used as loading control. In A and B mean+s.e.m. values of three independent experiments carried out in triplicate, in C mean+s.d. of three independent experiments are shown, while in D a representative result of three independent experiments is depicted. Statistical analysis was carried out by two-sided Student's t -test; * P-value
    Fluorescein Fragel Dna Fragmentation Detection Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 320 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc fragmented dna
    Altered timing affects the potency of NVP-BEZ235/doxorubicin combination therapy in SHEP NB cells. Three different treatment combinations were tested on SHEP NB cells, giving NVP-BEZ235 12 hrs prior to doxorubicin (Pre), giving both substances concurrently (Co), or giving NVP-BEZ235 12 hrs after the chemotherapeutic (Post). Importantly, the maximal incubation time with doxorubicin was kept constant at 24 hrs (earlier time points also shown in C and D). A SHEP NB cells were treated with NVP-BEZ235 and indicated concentrations of doxorubicin for 24 hrs, according to the scheme outlined above. <t>Apoptosis</t> was determined by FACS analysis of the <t>DNA</t> fragmentation of propidium iodide-stained nuclei, and percentage of specific DNA fragmentation is shown. B An alternative depiction of the data presented in A, highlighting the difference between the three NVP-BEZ235/doxorubicin combinations. For all following experiments 0.2 µg/ml doxorubicin was used. C Cells were either left untreated or treated as indicated and mitochondrial release of immunofluorescent-labeled cytochrome c was determined by FACS analysis. D Cells were either left untreated or treated as indicated. A Western blot analysis of caspase-3 processing served as surrogate read-out of caspase activation (appearance of the ∼12 kD cleavage fragment), β-actin was used as loading control. In A and B mean+s.e.m. values of three independent experiments carried out in triplicate, in C mean+s.d. of three independent experiments are shown, while in D a representative result of three independent experiments is depicted. Statistical analysis was carried out by two-sided Student's t -test; * P-value
    Fragmented Dna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 495 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim dna fragmentation
    Effect of bcl-2 overexpression on <t>Cdt-induced</t> G 2 arrest and <t>DNA</t> fragmentation. Jurkat neo and Jurkat bcl-2 cells were incubated in the presence of medium alone (Ctl), 0.05 ng/ml Cdt, or 5 ng/ml Cdt for 18 h; the cells were then stained with propidium iodide and by TUNEL to measure cell cycle distribution (panels on the right) and DNA fragmentation (panels on the left), respectively. The percentages of cells in the phases of the cell cycle are indicated, as are the percentages of cells exhibiting DNA fragmentation. The results are representative of at least three experiments.
    Dna Fragmentation, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs klenow fragment dna polymerase
    Effect of bcl-2 overexpression on <t>Cdt-induced</t> G 2 arrest and <t>DNA</t> fragmentation. Jurkat neo and Jurkat bcl-2 cells were incubated in the presence of medium alone (Ctl), 0.05 ng/ml Cdt, or 5 ng/ml Cdt for 18 h; the cells were then stained with propidium iodide and by TUNEL to measure cell cycle distribution (panels on the right) and DNA fragmentation (panels on the left), respectively. The percentages of cells in the phases of the cell cycle are indicated, as are the percentages of cells exhibiting DNA fragmentation. The results are representative of at least three experiments.
    Klenow Fragment Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pbr322 dna bsuri haeiii marker
    Effect of bcl-2 overexpression on <t>Cdt-induced</t> G 2 arrest and <t>DNA</t> fragmentation. Jurkat neo and Jurkat bcl-2 cells were incubated in the presence of medium alone (Ctl), 0.05 ng/ml Cdt, or 5 ng/ml Cdt for 18 h; the cells were then stained with propidium iodide and by TUNEL to measure cell cycle distribution (panels on the right) and DNA fragmentation (panels on the left), respectively. The percentages of cells in the phases of the cell cycle are indicated, as are the percentages of cells exhibiting DNA fragmentation. The results are representative of at least three experiments.
    Pbr322 Dna Bsuri Haeiii Marker, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher strings dna fragments
    Effect of bcl-2 overexpression on <t>Cdt-induced</t> G 2 arrest and <t>DNA</t> fragmentation. Jurkat neo and Jurkat bcl-2 cells were incubated in the presence of medium alone (Ctl), 0.05 ng/ml Cdt, or 5 ng/ml Cdt for 18 h; the cells were then stained with propidium iodide and by TUNEL to measure cell cycle distribution (panels on the right) and DNA fragmentation (panels on the left), respectively. The percentages of cells in the phases of the cell cycle are indicated, as are the percentages of cells exhibiting DNA fragmentation. The results are representative of at least three experiments.
    Strings Dna Fragments, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 269 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Trevigen dna fragmentation
    The effects of CLK2 on radiation sensitivity of CCD-18Lu cells. Cells were transfected with either vacant vector or myc-CLK2 . At 24 h post-transfection, cells were exposed to 0.05–2 Gy of γ-rays and cell viability was assayed 48 h later. A , the overexpression of Myc-CLK2 at 72 h post-transfection. B , the cell viability of CLK2 overexpressing CCD-18Lu cells to ionizing radiation was determined by MTT assay. C , cell proliferation of CLK2 overexpressing cells after irradiation. D , effect of CLK2 overexpression on 2 Gy-induced <t>apoptosis.</t> <t>DNA</t> fragmentation was detected with HT TiterTACS Assay Kit for the quantification of apoptosis. B–D , data represent mean ± S.D. ( n = 3) and were analyzed by the t test. Data showed a significant difference compared with vector control at the indicated dose (*, p
    Dna Fragmentation, supplied by Trevigen, used in various techniques. Bioz Stars score: 93/100, based on 260 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    (A-B) Schematics illustrating the procedure to generate the slo E366G and slo loxP alleles via ends-out homologous recombination (A) and method for molecular validation (B). The slo locus is shown. Grey boxes represent 5’ and 3’ untranslated regions, black boxes represent both constitutive and alternatively spliced coding exons. Region encompassed by targeting arms is denoted by the grey dashed box. Linearized recombinogenic arms (Arm 1 and Arm 2, separated by loxP and mini- white + sequences) were liberated from a genomic p[w25 . 2] insertion as described in Supplemental Ref. 2. Potential recombinants were initially identified by the presence of non-white eye colour due to the mini- white + marker, then validated through PCR via the strategy shown in (B). Validated recombinants are denoted by a PCR product of approximately 3.3 kb amplified from single-fly genomic DNA that was absent in non-recombinant control DNA (-ve control).

    Journal: bioRxiv

    Article Title: A knock-in Drosophila model supports a conserved link between potassium channelopathy and involuntary movement

    doi: 10.1101/2020.02.20.957571

    Figure Lengend Snippet: (A-B) Schematics illustrating the procedure to generate the slo E366G and slo loxP alleles via ends-out homologous recombination (A) and method for molecular validation (B). The slo locus is shown. Grey boxes represent 5’ and 3’ untranslated regions, black boxes represent both constitutive and alternatively spliced coding exons. Region encompassed by targeting arms is denoted by the grey dashed box. Linearized recombinogenic arms (Arm 1 and Arm 2, separated by loxP and mini- white + sequences) were liberated from a genomic p[w25 . 2] insertion as described in Supplemental Ref. 2. Potential recombinants were initially identified by the presence of non-white eye colour due to the mini- white + marker, then validated through PCR via the strategy shown in (B). Validated recombinants are denoted by a PCR product of approximately 3.3 kb amplified from single-fly genomic DNA that was absent in non-recombinant control DNA (-ve control).

    Article Snippet: The A1097G point mutation resulting in the E366G amino-acid change was introduced into Arm 2 via a customised DNA fragment (GeneArt Gene Synthesis, ThermoFi sher Scientific) that was used to replace the 5’ 722 bp of Arm 2.

    Techniques: Homologous Recombination, Marker, Polymerase Chain Reaction, Amplification, Recombinant

    Performance of various hgRNAs. ( A ) The sequence of seven different hgRNAs. Transcription start site (TSS), spacer sequence, and PAM are marked with grey, blue, and pink boxes, respectively. ( B,C,D ) Cas9 is induced in cell lines with the hgRNAs from panel A integrated genomically. DNA samples harvested before (t=0 days) and at various points after induction are characterized by high-throughput sequencing to quantify mutations, functionality, and generated diversity for each hgRNA. See also Supplementary table 1 . ( E ) Design of four longer variants of hgRNA-A21. Stuffer sequences of 5, 30, 55, or 80 base pairs were added upstream of a spacer very similar to the A21 spacer to obtain the four increasingly lengthy A’ variants. ( F,G,H ) Cas9 is induced in cell lines with the hgRNAs from panel E integrated genomically. DNA samples harvested before (t=0 days) and at various points after induction are characterized by high-throughput sequencing to quantify mutations and functionality of hgRNA loci. Data points represent duplicate experiments.

    Journal: Nature methods

    Article Title: Rapidly evolving homing CRISPR barcodes

    doi: 10.1038/nmeth.4108

    Figure Lengend Snippet: Performance of various hgRNAs. ( A ) The sequence of seven different hgRNAs. Transcription start site (TSS), spacer sequence, and PAM are marked with grey, blue, and pink boxes, respectively. ( B,C,D ) Cas9 is induced in cell lines with the hgRNAs from panel A integrated genomically. DNA samples harvested before (t=0 days) and at various points after induction are characterized by high-throughput sequencing to quantify mutations, functionality, and generated diversity for each hgRNA. See also Supplementary table 1 . ( E ) Design of four longer variants of hgRNA-A21. Stuffer sequences of 5, 30, 55, or 80 base pairs were added upstream of a spacer very similar to the A21 spacer to obtain the four increasingly lengthy A’ variants. ( F,G,H ) Cas9 is induced in cell lines with the hgRNAs from panel E integrated genomically. DNA samples harvested before (t=0 days) and at various points after induction are characterized by high-throughput sequencing to quantify mutations and functionality of hgRNA loci. Data points represent duplicate experiments.

    Article Snippet: The target locus of sgRNA-A21 and hgRNA-A21 were constructed by incorporating corresponding gBlock (IDT DNA) synthesized DNA fragment into the pLKO.1 lentiviral backbone (MISSION shRNA vectors via SIGMA) which was modified to carry Blasticidin resistance.

    Techniques: Sequencing, Next-Generation Sequencing, Generated

    ROS generation triggered by FPKc and ES. SW-480 (A) and HEK-293 (B) cells were treated with FPKc and ES, and the ROS levels were measured by flow cytometry after staining with DCFH-DA. SW-480 cells were pretreated with NAC (5 mM) for 1 h, then intracellular ROS generation (C), DNA damage (D), cell viability (E) and apoptosis (F) were detected.

    Journal: PLoS ONE

    Article Title: Investigating Migration Inhibition and Apoptotic Effects of Fomitopsis pinicola Chloroform Extract on Human Colorectal Cancer SW-480 Cells

    doi: 10.1371/journal.pone.0101303

    Figure Lengend Snippet: ROS generation triggered by FPKc and ES. SW-480 (A) and HEK-293 (B) cells were treated with FPKc and ES, and the ROS levels were measured by flow cytometry after staining with DCFH-DA. SW-480 cells were pretreated with NAC (5 mM) for 1 h, then intracellular ROS generation (C), DNA damage (D), cell viability (E) and apoptosis (F) were detected.

    Article Snippet: Flow cytometry analysis of DNA fragmentation The method to analyze DNA fragmentation was flow fluorocytometric detection of DNA hypoploidy after adding propidium iodide (PI; Sigma, St. Louis, USA) to the dying cells and permeabilizing them by freeze-thawing .

    Techniques: Flow Cytometry, Cytometry, Staining

    Effects of FPKc and ES on DNA fragmentation of SW-480 (A) and HEK-293 (B) cells. Both Cells were treated with FPKc and ES for 12(PI) and analyzed by flow cytometry.

    Journal: PLoS ONE

    Article Title: Investigating Migration Inhibition and Apoptotic Effects of Fomitopsis pinicola Chloroform Extract on Human Colorectal Cancer SW-480 Cells

    doi: 10.1371/journal.pone.0101303

    Figure Lengend Snippet: Effects of FPKc and ES on DNA fragmentation of SW-480 (A) and HEK-293 (B) cells. Both Cells were treated with FPKc and ES for 12(PI) and analyzed by flow cytometry.

    Article Snippet: Flow cytometry analysis of DNA fragmentation The method to analyze DNA fragmentation was flow fluorocytometric detection of DNA hypoploidy after adding propidium iodide (PI; Sigma, St. Louis, USA) to the dying cells and permeabilizing them by freeze-thawing .

    Techniques: Flow Cytometry, Cytometry

    Mutation detection in the T4_promoter fragment. Three polymorphisms were detected by dHPLC in the T4_promoter fragment and were verified by DNA sequencing. The four dHPLC graphs correspond to the wild-type sequence (A), SNPs T-441C (B), SNP A-271G (C) and SNP G-259C (D). The wild-type pattern is visible in all dHPLC graphs as a 1 peak trace. SNP T-441C is identified by an unequal height 2 peak trace (B), SNP A-271G is identified by an equal height 2 peak trace (C) and SNP G-259C is identified by a 3 peak trace (D). The positions of the sequence variants in the T4_promoter fragment identified by dHPLC were determined by DNA sequencing. All SNPs were present in the heterozygous state.

    Journal: PLoS ONE

    Article Title: Toll-Like Receptor 4 (TLR4) and Typhoid Fever in Vietnam

    doi: 10.1371/journal.pone.0004800

    Figure Lengend Snippet: Mutation detection in the T4_promoter fragment. Three polymorphisms were detected by dHPLC in the T4_promoter fragment and were verified by DNA sequencing. The four dHPLC graphs correspond to the wild-type sequence (A), SNPs T-441C (B), SNP A-271G (C) and SNP G-259C (D). The wild-type pattern is visible in all dHPLC graphs as a 1 peak trace. SNP T-441C is identified by an unequal height 2 peak trace (B), SNP A-271G is identified by an equal height 2 peak trace (C) and SNP G-259C is identified by a 3 peak trace (D). The positions of the sequence variants in the T4_promoter fragment identified by dHPLC were determined by DNA sequencing. All SNPs were present in the heterozygous state.

    Article Snippet: Mutation Detection by dHPLC Primers and amplicons were designed to detect mutations in TLR4 by dHPLC according to the recommendations described by the manufacturer (Wave® DNA Fragment Analysis System, Transgenomic, USA) ( ).

    Techniques: Mutagenesis, DNA Sequencing, Sequencing

    Notch1 and Notch3 are direct downstream targets of Dlx5 ( A ) ChIP-seq analysis revealed Dlx5 binding sequences in Notch3 (MACS p value: −10*LOG10 = 70.54) and Irs2 (MACS p value: −10*LOG10 = 76.57) loci as depicted in screenshots from UCSC Genome Browser. Arrows point to binding intervals in each locus. ( B ) Multiple Em for Motif Elicitation (MEME) shows that the majority of the Dlx5 binding consensus site belongs to the classic binding motif (TAATT) of the Dlx family. ( C ) Irs2 promoters from human, monkey, rat and mouse contain Dlx5 binding consensus. ( D ) results of ChIP-qPCR assay confirmed that Dlx5 binds to two enhancer elements at end of Notch1 gene. ( E ) ChIP-qPCR verified that Dlx5 binds to enhancer element located in Notch3 intron 2. ( F ) schematic diagram of Notch1 and Notch3 promoter/enhancer reporter plasmids. Luciferase assay confirmed that Dlx5, but not Dlx5 mutant lacking the DNA binding domain, activates Notch1 ( G ) and Notch3 ( H ) promoters upon binding to their respective enhancer elements.

    Journal: Oncotarget

    Article Title: The homeoprotein Dlx5 drives murine T-cell lymphomagenesis by directly transactivating Notch and upregulating Akt signaling

    doi: 10.18632/oncotarget.14784

    Figure Lengend Snippet: Notch1 and Notch3 are direct downstream targets of Dlx5 ( A ) ChIP-seq analysis revealed Dlx5 binding sequences in Notch3 (MACS p value: −10*LOG10 = 70.54) and Irs2 (MACS p value: −10*LOG10 = 76.57) loci as depicted in screenshots from UCSC Genome Browser. Arrows point to binding intervals in each locus. ( B ) Multiple Em for Motif Elicitation (MEME) shows that the majority of the Dlx5 binding consensus site belongs to the classic binding motif (TAATT) of the Dlx family. ( C ) Irs2 promoters from human, monkey, rat and mouse contain Dlx5 binding consensus. ( D ) results of ChIP-qPCR assay confirmed that Dlx5 binds to two enhancer elements at end of Notch1 gene. ( E ) ChIP-qPCR verified that Dlx5 binds to enhancer element located in Notch3 intron 2. ( F ) schematic diagram of Notch1 and Notch3 promoter/enhancer reporter plasmids. Luciferase assay confirmed that Dlx5, but not Dlx5 mutant lacking the DNA binding domain, activates Notch1 ( G ) and Notch3 ( H ) promoters upon binding to their respective enhancer elements.

    Article Snippet: Myc-Tag antibody, as well as IgG control, were used to immunoprecipitate Dlx5/chromatin complexes, and captured DNA fragments were analyzed by Illumina GA II to obtain whole-genome datasets (available at GEO #GSE83778).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Magnetic Cell Separation, Real-time Polymerase Chain Reaction, Luciferase, Mutagenesis

    Lck-Dlx5 lymphomas exhibit a molecular signature reminiscent of human T-ALL ( A ) RNA microarray heatmap demonstrating that compared to thymocytes from three WT mice, primary T-cell lymphomas from three Lck-Dlx5 mice exhibit upregulation ( Red ) of genes involved in Notch signaling, cell cycle progression, chromatin modification, calcium homeostasis, glucose, fatty acid and nucleotide metabolism, as well as classical Dlx5 target genes. Down-regulated genes ( Green ) are implicated in pro-apoptotic events ( Tnf , Fas , Xaf1 , INFb signaling-related genes), tumor suppression, and DNA damage repair. ( B ) compared to T-cell lymphomas from Lck-MyrAkt2 mice, Lck-Dlx5 lymphomas show upregulation of Notch1/3 and downregulation of Lats2 , Appl2 , Jak3 , Tet2 , and Tnik . Note that in the interests of journal space, the heatmap shown here includes only one sample from each group. The full heatmaps for all three samples examined per group can be found in Supplementary Figure 2C. ( C ) real-time PCR on three T-cell samples from WT mice (samples 1–3), 11 primary lymphomas from Lck-Dlx5 mice (samples 4-14: F86-785, -801, -793, -1149; F63-0, -1263, -1210; F47-0, -1247, -918; and F84-1063), and 7 lymphomas from Lck-MyrAkt2 mice (samples 15-21: F72-918, -2811, -3148, -3154; and F420-577, -1174, -1073), confirming unique upregulation of Notch signaling in Dlx5-driven tumors. Data = mean ± SEM. ( D ) immunoblot showing overexpression of Notch1, NIC1, Notch3, Hes1 and Myc in lymphoma lines (F63-0, F47-0, F47-918) from Lck-Dlx5 mice. Loss of Pten expression and corresponding high levels of phospho-Akt in Lck-Dlx5 -derived tumor cells are also shown, as are cyclin D1 levels. Dlx5 proteins have high molecular weight (a, b) and low molecular weight (c) forms. Notch proteins have full length ( F ) and cleaved (C) forms. *, non-specific bands. ( E ) H E and immunohistochemical staining of lymphoma invading lung of Lck-Dlx5 mouse. Note strong staining for Notch1, Notch3, Hes1, Myc and phospho-Akt in lymphoma. (F) summary of real-time PCR analysis of RNA from 15 pediatric T-ALL specimens showing expression levels of DLX5 , NOTCH1 and NOTCH3 .

    Journal: Oncotarget

    Article Title: The homeoprotein Dlx5 drives murine T-cell lymphomagenesis by directly transactivating Notch and upregulating Akt signaling

    doi: 10.18632/oncotarget.14784

    Figure Lengend Snippet: Lck-Dlx5 lymphomas exhibit a molecular signature reminiscent of human T-ALL ( A ) RNA microarray heatmap demonstrating that compared to thymocytes from three WT mice, primary T-cell lymphomas from three Lck-Dlx5 mice exhibit upregulation ( Red ) of genes involved in Notch signaling, cell cycle progression, chromatin modification, calcium homeostasis, glucose, fatty acid and nucleotide metabolism, as well as classical Dlx5 target genes. Down-regulated genes ( Green ) are implicated in pro-apoptotic events ( Tnf , Fas , Xaf1 , INFb signaling-related genes), tumor suppression, and DNA damage repair. ( B ) compared to T-cell lymphomas from Lck-MyrAkt2 mice, Lck-Dlx5 lymphomas show upregulation of Notch1/3 and downregulation of Lats2 , Appl2 , Jak3 , Tet2 , and Tnik . Note that in the interests of journal space, the heatmap shown here includes only one sample from each group. The full heatmaps for all three samples examined per group can be found in Supplementary Figure 2C. ( C ) real-time PCR on three T-cell samples from WT mice (samples 1–3), 11 primary lymphomas from Lck-Dlx5 mice (samples 4-14: F86-785, -801, -793, -1149; F63-0, -1263, -1210; F47-0, -1247, -918; and F84-1063), and 7 lymphomas from Lck-MyrAkt2 mice (samples 15-21: F72-918, -2811, -3148, -3154; and F420-577, -1174, -1073), confirming unique upregulation of Notch signaling in Dlx5-driven tumors. Data = mean ± SEM. ( D ) immunoblot showing overexpression of Notch1, NIC1, Notch3, Hes1 and Myc in lymphoma lines (F63-0, F47-0, F47-918) from Lck-Dlx5 mice. Loss of Pten expression and corresponding high levels of phospho-Akt in Lck-Dlx5 -derived tumor cells are also shown, as are cyclin D1 levels. Dlx5 proteins have high molecular weight (a, b) and low molecular weight (c) forms. Notch proteins have full length ( F ) and cleaved (C) forms. *, non-specific bands. ( E ) H E and immunohistochemical staining of lymphoma invading lung of Lck-Dlx5 mouse. Note strong staining for Notch1, Notch3, Hes1, Myc and phospho-Akt in lymphoma. (F) summary of real-time PCR analysis of RNA from 15 pediatric T-ALL specimens showing expression levels of DLX5 , NOTCH1 and NOTCH3 .

    Article Snippet: Myc-Tag antibody, as well as IgG control, were used to immunoprecipitate Dlx5/chromatin complexes, and captured DNA fragments were analyzed by Illumina GA II to obtain whole-genome datasets (available at GEO #GSE83778).

    Techniques: Microarray, Mouse Assay, Modification, Real-time Polymerase Chain Reaction, Over Expression, Expressing, Derivative Assay, Molecular Weight, Immunohistochemistry, Staining

    Akt and Notch signaling are essential for proliferation and survival of Lck-Dlx5 lymphoma cells ( A ) ectopic expression of Pten or dominant-negative mastermind ( DNMM ) inhibits survival of Lck-Dlx5 tumor cells as shown by MTS assay. Control was ectopically expressed Gfp . ( B ) immunoblot analysis depicting expression of exogenous DNMM - Gfp and Pten . ( C ) tumor growth of tail vein-injected Lck-Dlx5 lymphoma cells into NSG mice. Weights of tumor cell-infiltrated liver and spleen are shown. ( D ) flow cytometry showing that Notch inhibitors XXI or DAPT suppress cell cycle progression of Lck-Dlx5 tumor cells. ( E and F ) Notch inhibitors decrease Notch1, Notch3, Hes1 and c-Myc protein levels. Cells were harvested 24 h after beginning treatment with inhibitor. ( G and H ) Akt inhibitor GSK690693 alone triggers sub-G1 DNA condensation, caspase 3 activation, and Parp cleavage in Lck-Dlx5 tumor cells. Upregulation of phospho-Akt in GSK690693-treated cells is indicative of a feedback loop to Akt, although downstream effectors of Akt signaling, e.g., p-mTOR and p-p70S6k are downregulated [ 44 ]. ( I and J ) when cells were treated with GSK690693 plus Notch inhibitors XXI or DAPT, apoptosis was significantly increased, as assessed by flow cytometry and immunoblotting. *, residual Hes1 band due to previous blotting with Hes1 antibody. Significance: * p

    Journal: Oncotarget

    Article Title: The homeoprotein Dlx5 drives murine T-cell lymphomagenesis by directly transactivating Notch and upregulating Akt signaling

    doi: 10.18632/oncotarget.14784

    Figure Lengend Snippet: Akt and Notch signaling are essential for proliferation and survival of Lck-Dlx5 lymphoma cells ( A ) ectopic expression of Pten or dominant-negative mastermind ( DNMM ) inhibits survival of Lck-Dlx5 tumor cells as shown by MTS assay. Control was ectopically expressed Gfp . ( B ) immunoblot analysis depicting expression of exogenous DNMM - Gfp and Pten . ( C ) tumor growth of tail vein-injected Lck-Dlx5 lymphoma cells into NSG mice. Weights of tumor cell-infiltrated liver and spleen are shown. ( D ) flow cytometry showing that Notch inhibitors XXI or DAPT suppress cell cycle progression of Lck-Dlx5 tumor cells. ( E and F ) Notch inhibitors decrease Notch1, Notch3, Hes1 and c-Myc protein levels. Cells were harvested 24 h after beginning treatment with inhibitor. ( G and H ) Akt inhibitor GSK690693 alone triggers sub-G1 DNA condensation, caspase 3 activation, and Parp cleavage in Lck-Dlx5 tumor cells. Upregulation of phospho-Akt in GSK690693-treated cells is indicative of a feedback loop to Akt, although downstream effectors of Akt signaling, e.g., p-mTOR and p-p70S6k are downregulated [ 44 ]. ( I and J ) when cells were treated with GSK690693 plus Notch inhibitors XXI or DAPT, apoptosis was significantly increased, as assessed by flow cytometry and immunoblotting. *, residual Hes1 band due to previous blotting with Hes1 antibody. Significance: * p

    Article Snippet: Myc-Tag antibody, as well as IgG control, were used to immunoprecipitate Dlx5/chromatin complexes, and captured DNA fragments were analyzed by Illumina GA II to obtain whole-genome datasets (available at GEO #GSE83778).

    Techniques: Expressing, Dominant Negative Mutation, MTS Assay, Injection, Mouse Assay, Flow Cytometry, Cytometry, Activation Assay

    Dlx5 induces activation of Notch and Akt during T-cell development Real-time PCR was performed on thymic T cells from 5-week-old Lck-Dlx5 mice (D1, D2, D3) prior to tumor development, WT littermates (W1,W2,W3), a 5-week-old Lck-MyrAkt2 mouse (Akt), and Lck-Dlx5 lymphomas (801, 793) to compare expression levels of full-length Notch1/3 ( A ) and Irs2 ( B ). ( C ) immunoblotting confirming increased expression of Notch1/3 in the non-malignant T cells from Lck-Dlx5 mice, with further enhanced expression in tumors from these mice. ( D and E ) CCC assay showing frequency of interaction of Dlx5-bound Notch1 enhancers to Notch1 promoter (D) and Dlx5-bound Notch3 enhancer to Notch3 promoter (E). N1-20K and N3-20K refer to control DNA segments 20 Kb downstream of Notch1 or Notch3 loci, respectively. ( F and G ) Flow cytometry analysis of various stages of T-cell development showing elevated expression of Notch1 (F) and Notch3 (G) in all DN stages (DN1-DN4) in Lck-Dlx5 mice compared to that of WT mice. ( H ) Flow cytometry analysis revealing increased expression of activated Akt kinase (p-Akt) in all DN stages in T cells from Lck-Dlx5 mice.

    Journal: Oncotarget

    Article Title: The homeoprotein Dlx5 drives murine T-cell lymphomagenesis by directly transactivating Notch and upregulating Akt signaling

    doi: 10.18632/oncotarget.14784

    Figure Lengend Snippet: Dlx5 induces activation of Notch and Akt during T-cell development Real-time PCR was performed on thymic T cells from 5-week-old Lck-Dlx5 mice (D1, D2, D3) prior to tumor development, WT littermates (W1,W2,W3), a 5-week-old Lck-MyrAkt2 mouse (Akt), and Lck-Dlx5 lymphomas (801, 793) to compare expression levels of full-length Notch1/3 ( A ) and Irs2 ( B ). ( C ) immunoblotting confirming increased expression of Notch1/3 in the non-malignant T cells from Lck-Dlx5 mice, with further enhanced expression in tumors from these mice. ( D and E ) CCC assay showing frequency of interaction of Dlx5-bound Notch1 enhancers to Notch1 promoter (D) and Dlx5-bound Notch3 enhancer to Notch3 promoter (E). N1-20K and N3-20K refer to control DNA segments 20 Kb downstream of Notch1 or Notch3 loci, respectively. ( F and G ) Flow cytometry analysis of various stages of T-cell development showing elevated expression of Notch1 (F) and Notch3 (G) in all DN stages (DN1-DN4) in Lck-Dlx5 mice compared to that of WT mice. ( H ) Flow cytometry analysis revealing increased expression of activated Akt kinase (p-Akt) in all DN stages in T cells from Lck-Dlx5 mice.

    Article Snippet: Myc-Tag antibody, as well as IgG control, were used to immunoprecipitate Dlx5/chromatin complexes, and captured DNA fragments were analyzed by Illumina GA II to obtain whole-genome datasets (available at GEO #GSE83778).

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Mouse Assay, Expressing, Countercurrent Chromatography, Flow Cytometry, Cytometry

    Altered timing affects the potency of NVP-BEZ235/doxorubicin combination therapy in SHEP NB cells. Three different treatment combinations were tested on SHEP NB cells, giving NVP-BEZ235 12 hrs prior to doxorubicin (Pre), giving both substances concurrently (Co), or giving NVP-BEZ235 12 hrs after the chemotherapeutic (Post). Importantly, the maximal incubation time with doxorubicin was kept constant at 24 hrs (earlier time points also shown in C and D). A SHEP NB cells were treated with NVP-BEZ235 and indicated concentrations of doxorubicin for 24 hrs, according to the scheme outlined above. Apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei, and percentage of specific DNA fragmentation is shown. B An alternative depiction of the data presented in A, highlighting the difference between the three NVP-BEZ235/doxorubicin combinations. For all following experiments 0.2 µg/ml doxorubicin was used. C Cells were either left untreated or treated as indicated and mitochondrial release of immunofluorescent-labeled cytochrome c was determined by FACS analysis. D Cells were either left untreated or treated as indicated. A Western blot analysis of caspase-3 processing served as surrogate read-out of caspase activation (appearance of the ∼12 kD cleavage fragment), β-actin was used as loading control. In A and B mean+s.e.m. values of three independent experiments carried out in triplicate, in C mean+s.d. of three independent experiments are shown, while in D a representative result of three independent experiments is depicted. Statistical analysis was carried out by two-sided Student's t -test; * P-value

    Journal: PLoS ONE

    Article Title: Sequential Dosing in Chemosensitization: Targeting the PI3K/Akt/mTOR Pathway in Neuroblastoma

    doi: 10.1371/journal.pone.0083128

    Figure Lengend Snippet: Altered timing affects the potency of NVP-BEZ235/doxorubicin combination therapy in SHEP NB cells. Three different treatment combinations were tested on SHEP NB cells, giving NVP-BEZ235 12 hrs prior to doxorubicin (Pre), giving both substances concurrently (Co), or giving NVP-BEZ235 12 hrs after the chemotherapeutic (Post). Importantly, the maximal incubation time with doxorubicin was kept constant at 24 hrs (earlier time points also shown in C and D). A SHEP NB cells were treated with NVP-BEZ235 and indicated concentrations of doxorubicin for 24 hrs, according to the scheme outlined above. Apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei, and percentage of specific DNA fragmentation is shown. B An alternative depiction of the data presented in A, highlighting the difference between the three NVP-BEZ235/doxorubicin combinations. For all following experiments 0.2 µg/ml doxorubicin was used. C Cells were either left untreated or treated as indicated and mitochondrial release of immunofluorescent-labeled cytochrome c was determined by FACS analysis. D Cells were either left untreated or treated as indicated. A Western blot analysis of caspase-3 processing served as surrogate read-out of caspase activation (appearance of the ∼12 kD cleavage fragment), β-actin was used as loading control. In A and B mean+s.e.m. values of three independent experiments carried out in triplicate, in C mean+s.d. of three independent experiments are shown, while in D a representative result of three independent experiments is depicted. Statistical analysis was carried out by two-sided Student's t -test; * P-value

    Article Snippet: Cell death measurement The cell death readout was DNA fragmentation (sub-G1 peak), a hallmark of apoptosis, as assessed by fluorescence-activated cell-sorting (FACScan, BD Bioscience) analysis of DNA fragmentation of propidium iodide-stained nuclei as previously described .

    Techniques: Incubation, FACS, Staining, Labeling, Western Blot, Activation Assay

    Sensitization for doxorubicin-induced apoptosis via posttreatment with NVP-BEZ235 is mediated via VDAC1. A SHEP NB cells were treated for 12.5-BEZ235 for the last 0.5 hr. Either Bim, Bax or Bad was then immunoprecipitated and interaction partners that are phosphorylated on Serine or Threonine were visualized by Western blot analysis. A ∼30 kD protein, the presence of which appears to depend on NVP-BEZ235 addition, was identified as VDAC by VDAC1/Porin-specific antibody. IgG H – heavy chain. B Cells were left untreated, treated for 12.5 hrs with Doxorubicin, or after 12 hrs for 0.5 hr with NVP-BEZ235, or a combination of both (first 12 hrs with Doxorubicin alone, followed by the addition of NVP-BEZ235 for 0.5 hr). VDAC was immunoprecipitated and its phosphorylation status was probed. IgG L – light chain. C Cells were left untreated, treated for 12.5 hrs with doxorubicin, or after 12 hrs for 0.5 hr with NVP-BEZ235, or a combination of both (first 12 hrs with doxorubicin alone, followed by the addition of NVP-BEZ235 for 0.5 hr). Protein expression levels and phosphorylation status of GSK3β were analyzed by Western blotting, GAPDH served as loading control. D Cells were treated either for 12.5 hrs with doxorubicin, or a combination of doxorubicin and NVP-BEZ235, (first 12 hrs with doxorubicin alone, followed by the addition of NVP-BEZ235 for 0.5 hr). This was followed by immunoprecipitation of GSK3β and analysis of this protein's interaction with VDAC via immunoblotting. E Cells were again treated with a combination of doxorubicin and NVP-BEZ235, (first 12 hrs with doxorubicin alone, followed by the addition of NVP-BEZ235 for 0.5 hr), during the last hour in the absence or presence of the GSK3β-specific inhibitor SB415286. This was followed by immunoprecipitation of GSK3β and analysis of this protein's interaction with VDAC. F Apoptosis in cells treated for 24 hrs with doxorubicin, for 12.5 hrs with SB415286, for 12 hrs with NVP-BEZ235, or a combination of those substances was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei, and percentage of specific DNA fragmentation is shown. Shown in A to E are representative blots of at least two independent experiments, in F the mean+s.e.m. of three independent experiments performed in triplicate is depicted. Statistical analysis was carried out by two-sided Student's t -test; * P-value

    Journal: PLoS ONE

    Article Title: Sequential Dosing in Chemosensitization: Targeting the PI3K/Akt/mTOR Pathway in Neuroblastoma

    doi: 10.1371/journal.pone.0083128

    Figure Lengend Snippet: Sensitization for doxorubicin-induced apoptosis via posttreatment with NVP-BEZ235 is mediated via VDAC1. A SHEP NB cells were treated for 12.5-BEZ235 for the last 0.5 hr. Either Bim, Bax or Bad was then immunoprecipitated and interaction partners that are phosphorylated on Serine or Threonine were visualized by Western blot analysis. A ∼30 kD protein, the presence of which appears to depend on NVP-BEZ235 addition, was identified as VDAC by VDAC1/Porin-specific antibody. IgG H – heavy chain. B Cells were left untreated, treated for 12.5 hrs with Doxorubicin, or after 12 hrs for 0.5 hr with NVP-BEZ235, or a combination of both (first 12 hrs with Doxorubicin alone, followed by the addition of NVP-BEZ235 for 0.5 hr). VDAC was immunoprecipitated and its phosphorylation status was probed. IgG L – light chain. C Cells were left untreated, treated for 12.5 hrs with doxorubicin, or after 12 hrs for 0.5 hr with NVP-BEZ235, or a combination of both (first 12 hrs with doxorubicin alone, followed by the addition of NVP-BEZ235 for 0.5 hr). Protein expression levels and phosphorylation status of GSK3β were analyzed by Western blotting, GAPDH served as loading control. D Cells were treated either for 12.5 hrs with doxorubicin, or a combination of doxorubicin and NVP-BEZ235, (first 12 hrs with doxorubicin alone, followed by the addition of NVP-BEZ235 for 0.5 hr). This was followed by immunoprecipitation of GSK3β and analysis of this protein's interaction with VDAC via immunoblotting. E Cells were again treated with a combination of doxorubicin and NVP-BEZ235, (first 12 hrs with doxorubicin alone, followed by the addition of NVP-BEZ235 for 0.5 hr), during the last hour in the absence or presence of the GSK3β-specific inhibitor SB415286. This was followed by immunoprecipitation of GSK3β and analysis of this protein's interaction with VDAC. F Apoptosis in cells treated for 24 hrs with doxorubicin, for 12.5 hrs with SB415286, for 12 hrs with NVP-BEZ235, or a combination of those substances was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei, and percentage of specific DNA fragmentation is shown. Shown in A to E are representative blots of at least two independent experiments, in F the mean+s.e.m. of three independent experiments performed in triplicate is depicted. Statistical analysis was carried out by two-sided Student's t -test; * P-value

    Article Snippet: Cell death measurement The cell death readout was DNA fragmentation (sub-G1 peak), a hallmark of apoptosis, as assessed by fluorescence-activated cell-sorting (FACScan, BD Bioscience) analysis of DNA fragmentation of propidium iodide-stained nuclei as previously described .

    Techniques: Immunoprecipitation, Western Blot, Expressing, FACS, Staining

    The superiority of posttreatment with NVP-BEZ235 is not restricted to one NB cell line and doxorubicin. A SH-SY5Y NB cells were treated as indicated by the scheme and apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. B Kelly NB cells were treated as indicated by the scheme and apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. C SHEP NB cells were treated as indicated by the scheme, substituting doxorubicin with either 1.0 µg/ml Cisplatin (Cis), 0.03 µg/ml Topotecan (Topo) or 5.0 µg/ml Etoposide (VP16). Apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. D D54 glioblastoma cells were treated as indicated by the scheme and apoptosis after doxorubicin (0.3 µg/ml doxorubicin) treatment was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. In A to D mean+s.e.m. values of three independent experiments carried out in triplicate are shown. Statistical analysis was carried out by two-sided Student's t -test; * P-value

    Journal: PLoS ONE

    Article Title: Sequential Dosing in Chemosensitization: Targeting the PI3K/Akt/mTOR Pathway in Neuroblastoma

    doi: 10.1371/journal.pone.0083128

    Figure Lengend Snippet: The superiority of posttreatment with NVP-BEZ235 is not restricted to one NB cell line and doxorubicin. A SH-SY5Y NB cells were treated as indicated by the scheme and apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. B Kelly NB cells were treated as indicated by the scheme and apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. C SHEP NB cells were treated as indicated by the scheme, substituting doxorubicin with either 1.0 µg/ml Cisplatin (Cis), 0.03 µg/ml Topotecan (Topo) or 5.0 µg/ml Etoposide (VP16). Apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. D D54 glioblastoma cells were treated as indicated by the scheme and apoptosis after doxorubicin (0.3 µg/ml doxorubicin) treatment was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. In A to D mean+s.e.m. values of three independent experiments carried out in triplicate are shown. Statistical analysis was carried out by two-sided Student's t -test; * P-value

    Article Snippet: Cell death measurement The cell death readout was DNA fragmentation (sub-G1 peak), a hallmark of apoptosis, as assessed by fluorescence-activated cell-sorting (FACScan, BD Bioscience) analysis of DNA fragmentation of propidium iodide-stained nuclei as previously described .

    Techniques: FACS, Staining

    The effects of the PI3K/mTOR inhibitor NVP-BEZ235 on SHEP NB cells. A Cells were either left untreated, treated for 24 µM PI-103, a well-characterized pan-PI3K inhibitor used as positive control, or the indicated concentrations of NVP-BEZ235. Protein expression levels and phosphorylation status of Akt and S6 ribosomal protein served as surrogate read-outs for PI3K and mTOR activity, respectively, and were analyzed by Western blotting, β-actin served as loading control. B Cells were either left untreated, treated for 24 hrs with either 0.6 µM PI-103 as positive control, or 0.6 µM NVP-BEZ235 for the indicated lengths of time. Protein expression levels and phosphorylation status of Akt and S6 ribosomal protein were analyzed by Western blotting, β-actin served as loading control. C Cells were either left untreated, treated for indicated length of time with 0.6 µM NVP-BEZ235, or treated for 24 hrs with 0.2 µg/ml doxorubicin as positive control. Protein expression levels and cleavage of caspase-.3 protein were analyzed by Western blotting, β-actin served as loading control. D Cells were cultured either in the presence or absence of 0.6 µM of NVP-BEZ235 for 24, 48 and 72 hrs, followed by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. The percentage of absolute DNA fragmentation is shown as readout of apoptosis. E 24, 48 and 72 hrs after treatment with 0.6 µM NVP-BEZ235 total cell numbers of treated and untreated cells were counted. F Cell cycle distribution (untreated control and samples treated with 0.6 µM of NVP-BEZ235) was determined after indicated times by FACS analysis of propidium iodide-stained nuclei. G Either untreated controls, or cells treated for 12 and 24 hrs with 0.6 µM NVP-BEZ235 were stained for Ki67 protein expression and evaluated by immunofluorescent microscopy. In A–C and F a representative result of two independent experiments is depicted, while in D and E mean+s.e.m. values of at least three independent experiments carried out in triplicate are shown. Shown in F is the mean of three independent experiments carried out in triplicate, in G the mean+SD of three independent experiments. Statistical analysis was carried out by two-sided Student's t -test; * P-value

    Journal: PLoS ONE

    Article Title: Sequential Dosing in Chemosensitization: Targeting the PI3K/Akt/mTOR Pathway in Neuroblastoma

    doi: 10.1371/journal.pone.0083128

    Figure Lengend Snippet: The effects of the PI3K/mTOR inhibitor NVP-BEZ235 on SHEP NB cells. A Cells were either left untreated, treated for 24 µM PI-103, a well-characterized pan-PI3K inhibitor used as positive control, or the indicated concentrations of NVP-BEZ235. Protein expression levels and phosphorylation status of Akt and S6 ribosomal protein served as surrogate read-outs for PI3K and mTOR activity, respectively, and were analyzed by Western blotting, β-actin served as loading control. B Cells were either left untreated, treated for 24 hrs with either 0.6 µM PI-103 as positive control, or 0.6 µM NVP-BEZ235 for the indicated lengths of time. Protein expression levels and phosphorylation status of Akt and S6 ribosomal protein were analyzed by Western blotting, β-actin served as loading control. C Cells were either left untreated, treated for indicated length of time with 0.6 µM NVP-BEZ235, or treated for 24 hrs with 0.2 µg/ml doxorubicin as positive control. Protein expression levels and cleavage of caspase-.3 protein were analyzed by Western blotting, β-actin served as loading control. D Cells were cultured either in the presence or absence of 0.6 µM of NVP-BEZ235 for 24, 48 and 72 hrs, followed by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. The percentage of absolute DNA fragmentation is shown as readout of apoptosis. E 24, 48 and 72 hrs after treatment with 0.6 µM NVP-BEZ235 total cell numbers of treated and untreated cells were counted. F Cell cycle distribution (untreated control and samples treated with 0.6 µM of NVP-BEZ235) was determined after indicated times by FACS analysis of propidium iodide-stained nuclei. G Either untreated controls, or cells treated for 12 and 24 hrs with 0.6 µM NVP-BEZ235 were stained for Ki67 protein expression and evaluated by immunofluorescent microscopy. In A–C and F a representative result of two independent experiments is depicted, while in D and E mean+s.e.m. values of at least three independent experiments carried out in triplicate are shown. Shown in F is the mean of three independent experiments carried out in triplicate, in G the mean+SD of three independent experiments. Statistical analysis was carried out by two-sided Student's t -test; * P-value

    Article Snippet: Cell death measurement The cell death readout was DNA fragmentation (sub-G1 peak), a hallmark of apoptosis, as assessed by fluorescence-activated cell-sorting (FACScan, BD Bioscience) analysis of DNA fragmentation of propidium iodide-stained nuclei as previously described .

    Techniques: Positive Control, Expressing, Activity Assay, Western Blot, Cell Culture, FACS, Staining, Microscopy

    The effect of several inhibitors of PI3K/mTOR signaling on SHEP NB cell survival. A SHEP NB cells were treated with doxorubicin for 24(control), or in the presence of the indicated pharmacological inhibitor, which was given 12 hrs prior to doxorubicin (Pre), or simultaneously with doxorubicin (Co), or 12 hrs after doxorubicin (Post). Apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. B Comparison of different treatment strategies, either using the pharmacolgical inhibitors as single agents or in combination. The sensitization effect is depicted as X-fold increase in cell death (as determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei) over treatment with doxorubicin alone. C Cells were either left untreated or treated with either NVP-BEZ235 posttreatment, or the complex combination therapy shown to work best in B, in the presence or absence of doxorubicin. Cells were treated with Nu7026 for 24.5 hrs, with doxorubicin and/or rapamycin for 12.5 hrs, 0.5 hrs with NVP-BEZ235 and allowed to grow 10 days. In A and B mean+s.e.m. values of three independent experiments carried out in triplicate are shown, in C a representative result of two independent experiments is depicted. Statistical analysis was carried out by two-sided Student's t -test; * P-value

    Journal: PLoS ONE

    Article Title: Sequential Dosing in Chemosensitization: Targeting the PI3K/Akt/mTOR Pathway in Neuroblastoma

    doi: 10.1371/journal.pone.0083128

    Figure Lengend Snippet: The effect of several inhibitors of PI3K/mTOR signaling on SHEP NB cell survival. A SHEP NB cells were treated with doxorubicin for 24(control), or in the presence of the indicated pharmacological inhibitor, which was given 12 hrs prior to doxorubicin (Pre), or simultaneously with doxorubicin (Co), or 12 hrs after doxorubicin (Post). Apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. B Comparison of different treatment strategies, either using the pharmacolgical inhibitors as single agents or in combination. The sensitization effect is depicted as X-fold increase in cell death (as determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei) over treatment with doxorubicin alone. C Cells were either left untreated or treated with either NVP-BEZ235 posttreatment, or the complex combination therapy shown to work best in B, in the presence or absence of doxorubicin. Cells were treated with Nu7026 for 24.5 hrs, with doxorubicin and/or rapamycin for 12.5 hrs, 0.5 hrs with NVP-BEZ235 and allowed to grow 10 days. In A and B mean+s.e.m. values of three independent experiments carried out in triplicate are shown, in C a representative result of two independent experiments is depicted. Statistical analysis was carried out by two-sided Student's t -test; * P-value

    Article Snippet: Cell death measurement The cell death readout was DNA fragmentation (sub-G1 peak), a hallmark of apoptosis, as assessed by fluorescence-activated cell-sorting (FACScan, BD Bioscience) analysis of DNA fragmentation of propidium iodide-stained nuclei as previously described .

    Techniques: FACS, Staining

    Apoptosis sensitization occurs at the mitochondrial level. A SHEP NB cells were either left untreated (control) or treated as indicated, followed by a Western blot analysis of the caspase-3 processing kinetic, with β-actin as loading control. B The loss of mitochondrial membrane potential (MMP) was analyzed after indicated treatment. Cells were incubated with TMRM dye prior to FACS analysis. C Cells were either left untreated or treated as indicated. The DNA damage was assayed by single cell gel electrophoresis (Comet) assay and expressed as Mean Olive Tail Moment. D Cells were treated for the indicated length of time with 0.6 µM NVP-BEZ235, 0.2 µg/ml doxorubicin, 20 nM Bafilomycin A1 (a inhibitor of the late stages of autophagy that blocks fusion between autophagosomes and lysosomes), or combinations thereof. The percentage of autophagic cells was then determined by counting cells with LC3 foci. In A representative results of two independent experiments are shown, in B mean+s.e.m. of three independent experiments carried out in triplicate are shown, while in C mean+s.d. of two independent experiments are depicted. In D mean+s.d. Of three independent experiments are depicted. Statistical analysis was carried out by two-sided Student's t -test; * P-value

    Journal: PLoS ONE

    Article Title: Sequential Dosing in Chemosensitization: Targeting the PI3K/Akt/mTOR Pathway in Neuroblastoma

    doi: 10.1371/journal.pone.0083128

    Figure Lengend Snippet: Apoptosis sensitization occurs at the mitochondrial level. A SHEP NB cells were either left untreated (control) or treated as indicated, followed by a Western blot analysis of the caspase-3 processing kinetic, with β-actin as loading control. B The loss of mitochondrial membrane potential (MMP) was analyzed after indicated treatment. Cells were incubated with TMRM dye prior to FACS analysis. C Cells were either left untreated or treated as indicated. The DNA damage was assayed by single cell gel electrophoresis (Comet) assay and expressed as Mean Olive Tail Moment. D Cells were treated for the indicated length of time with 0.6 µM NVP-BEZ235, 0.2 µg/ml doxorubicin, 20 nM Bafilomycin A1 (a inhibitor of the late stages of autophagy that blocks fusion between autophagosomes and lysosomes), or combinations thereof. The percentage of autophagic cells was then determined by counting cells with LC3 foci. In A representative results of two independent experiments are shown, in B mean+s.e.m. of three independent experiments carried out in triplicate are shown, while in C mean+s.d. of two independent experiments are depicted. In D mean+s.d. Of three independent experiments are depicted. Statistical analysis was carried out by two-sided Student's t -test; * P-value

    Article Snippet: Cell death measurement The cell death readout was DNA fragmentation (sub-G1 peak), a hallmark of apoptosis, as assessed by fluorescence-activated cell-sorting (FACScan, BD Bioscience) analysis of DNA fragmentation of propidium iodide-stained nuclei as previously described .

    Techniques: Western Blot, Incubation, FACS, Single Cell Gel Electrophoresis

    Effect of bcl-2 overexpression on Cdt-induced G 2 arrest and DNA fragmentation. Jurkat neo and Jurkat bcl-2 cells were incubated in the presence of medium alone (Ctl), 0.05 ng/ml Cdt, or 5 ng/ml Cdt for 18 h; the cells were then stained with propidium iodide and by TUNEL to measure cell cycle distribution (panels on the right) and DNA fragmentation (panels on the left), respectively. The percentages of cells in the phases of the cell cycle are indicated, as are the percentages of cells exhibiting DNA fragmentation. The results are representative of at least three experiments.

    Journal: Infection and Immunity

    Article Title: Exposure of Lymphocytes to High Doses of Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Induces Rapid Onset of Apoptosis-Mediated DNA Fragmentation

    doi: 10.1128/IAI.74.4.2080-2092.2006

    Figure Lengend Snippet: Effect of bcl-2 overexpression on Cdt-induced G 2 arrest and DNA fragmentation. Jurkat neo and Jurkat bcl-2 cells were incubated in the presence of medium alone (Ctl), 0.05 ng/ml Cdt, or 5 ng/ml Cdt for 18 h; the cells were then stained with propidium iodide and by TUNEL to measure cell cycle distribution (panels on the right) and DNA fragmentation (panels on the left), respectively. The percentages of cells in the phases of the cell cycle are indicated, as are the percentages of cells exhibiting DNA fragmentation. The results are representative of at least three experiments.

    Article Snippet: DNA fragmentation in Cdt-treated Jurkat cells was measured using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay (in situ cell death detection kit; Boehringer Mannheim, Indianapolis, IN) in conjunction with Hoechst 33342 to measure cell cycle progression.

    Techniques: Over Expression, Incubation, CTL Assay, Staining, TUNEL Assay

    Comparison of the effects of Cdt on cell cycle arrest, BrdU incorporation, and DNA fragmentation. Jurkat cells were treated with medium (control) or a low dose (0.05 ng/ml) or high dose (5 ng/ml) of Cdt for 18 h. The cells were then analyzed by flow cytometry to determine the cell cycle distribution using propidium iodide (A) and BrdU incorporation in order to directly assess the percentage of cells in the S phase (B) and DNA fragmentation (TUNEL) along with the cell cycle (Hoechst fluorescence) (C). The percentages of cells in the phases of the cell cycle (based on propidium iodide fluorescence) are shown in panel A. The percentages of S-phase cells determined by BrdU incorporation are indicated in panel B; cells that exhibited fluorescence greater than the fluorescence observed in controls (indicated by a line) were considered to be positive. The percentages of cells exhibiting DNA fragmentation are indicated in panel C; fluorescence values greater than the fluorescence observed in cells treated with the control antibody (indicated by a line) were considered to be positive. The results are representative of at least three experiments.

    Journal: Infection and Immunity

    Article Title: Exposure of Lymphocytes to High Doses of Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Induces Rapid Onset of Apoptosis-Mediated DNA Fragmentation

    doi: 10.1128/IAI.74.4.2080-2092.2006

    Figure Lengend Snippet: Comparison of the effects of Cdt on cell cycle arrest, BrdU incorporation, and DNA fragmentation. Jurkat cells were treated with medium (control) or a low dose (0.05 ng/ml) or high dose (5 ng/ml) of Cdt for 18 h. The cells were then analyzed by flow cytometry to determine the cell cycle distribution using propidium iodide (A) and BrdU incorporation in order to directly assess the percentage of cells in the S phase (B) and DNA fragmentation (TUNEL) along with the cell cycle (Hoechst fluorescence) (C). The percentages of cells in the phases of the cell cycle (based on propidium iodide fluorescence) are shown in panel A. The percentages of S-phase cells determined by BrdU incorporation are indicated in panel B; cells that exhibited fluorescence greater than the fluorescence observed in controls (indicated by a line) were considered to be positive. The percentages of cells exhibiting DNA fragmentation are indicated in panel C; fluorescence values greater than the fluorescence observed in cells treated with the control antibody (indicated by a line) were considered to be positive. The results are representative of at least three experiments.

    Article Snippet: DNA fragmentation in Cdt-treated Jurkat cells was measured using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay (in situ cell death detection kit; Boehringer Mannheim, Indianapolis, IN) in conjunction with Hoechst 33342 to measure cell cycle progression.

    Techniques: BrdU Incorporation Assay, Flow Cytometry, Cytometry, TUNEL Assay, Fluorescence

    Effect of zvad on Cdt-induced G 2 arrest and DNA fragmentation. Jurkat cells were pretreated with 50 μM zvad or medium for 30 min; this was followed by addition of either medium (Ctl) or Cdt. Cells were incubated for 18 h and then analyzed for DNA fragmentation (panels on the left) and cell cycle distribution (panels on the right) as described in Materials and Methods. The percentages of cells in the phases of the cell cycle are indicated, as are the percentages of cells exhibiting DNA fragmentation; the regions where there was positive dUTP-FITC fluorescence are indicated by the bars, and the results were based upon control samples.

    Journal: Infection and Immunity

    Article Title: Exposure of Lymphocytes to High Doses of Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Induces Rapid Onset of Apoptosis-Mediated DNA Fragmentation

    doi: 10.1128/IAI.74.4.2080-2092.2006

    Figure Lengend Snippet: Effect of zvad on Cdt-induced G 2 arrest and DNA fragmentation. Jurkat cells were pretreated with 50 μM zvad or medium for 30 min; this was followed by addition of either medium (Ctl) or Cdt. Cells were incubated for 18 h and then analyzed for DNA fragmentation (panels on the left) and cell cycle distribution (panels on the right) as described in Materials and Methods. The percentages of cells in the phases of the cell cycle are indicated, as are the percentages of cells exhibiting DNA fragmentation; the regions where there was positive dUTP-FITC fluorescence are indicated by the bars, and the results were based upon control samples.

    Article Snippet: DNA fragmentation in Cdt-treated Jurkat cells was measured using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay (in situ cell death detection kit; Boehringer Mannheim, Indianapolis, IN) in conjunction with Hoechst 33342 to measure cell cycle progression.

    Techniques: CTL Assay, Incubation, Fluorescence

    Effect of Cdt holotoxin on lymphocyte G 2 arrest and DNA fragmentation. Jurkat cells were treated with various concentrations of Cdt holotoxin for 18 h. The cells were then analyzed by flow cytometry to determine both the cell cycle distribution and the presence of DNA fragmentation as described in Methods and Materials. The percentage of G 2 cells (•) and the percentage of cells exhibiting DNA fragmentation (▪) are plotted versus Cdt concentration; the data are means ± standard deviations for three experiments. For control cells exposed to medium 9.1% of the cells were in the G 2 phase; 4.9% of the control cells exhibited DNA fragmentation.

    Journal: Infection and Immunity

    Article Title: Exposure of Lymphocytes to High Doses of Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Induces Rapid Onset of Apoptosis-Mediated DNA Fragmentation

    doi: 10.1128/IAI.74.4.2080-2092.2006

    Figure Lengend Snippet: Effect of Cdt holotoxin on lymphocyte G 2 arrest and DNA fragmentation. Jurkat cells were treated with various concentrations of Cdt holotoxin for 18 h. The cells were then analyzed by flow cytometry to determine both the cell cycle distribution and the presence of DNA fragmentation as described in Methods and Materials. The percentage of G 2 cells (•) and the percentage of cells exhibiting DNA fragmentation (▪) are plotted versus Cdt concentration; the data are means ± standard deviations for three experiments. For control cells exposed to medium 9.1% of the cells were in the G 2 phase; 4.9% of the control cells exhibited DNA fragmentation.

    Article Snippet: DNA fragmentation in Cdt-treated Jurkat cells was measured using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay (in situ cell death detection kit; Boehringer Mannheim, Indianapolis, IN) in conjunction with Hoechst 33342 to measure cell cycle progression.

    Techniques: Flow Cytometry, Cytometry, Concentration Assay

    Kinetics of Cdt-induced DNA degradation. Jurkat cells were treated with Cdt or medium for 24, 48, or 72 h. The cells were then assessed for DNA fragmentation using the TUNEL assay and FACS. The bars indicate regions where there was positive fluorescence; the percentages of cells in these regions that exhibited DNA fragmentation are indicated, and the degrees of fragmentation are indicated by the MCF values. The results are representative of three experiments.

    Journal: Infection and Immunity

    Article Title: Exposure of Lymphocytes to High Doses of Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Induces Rapid Onset of Apoptosis-Mediated DNA Fragmentation

    doi: 10.1128/IAI.74.4.2080-2092.2006

    Figure Lengend Snippet: Kinetics of Cdt-induced DNA degradation. Jurkat cells were treated with Cdt or medium for 24, 48, or 72 h. The cells were then assessed for DNA fragmentation using the TUNEL assay and FACS. The bars indicate regions where there was positive fluorescence; the percentages of cells in these regions that exhibited DNA fragmentation are indicated, and the degrees of fragmentation are indicated by the MCF values. The results are representative of three experiments.

    Article Snippet: DNA fragmentation in Cdt-treated Jurkat cells was measured using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay (in situ cell death detection kit; Boehringer Mannheim, Indianapolis, IN) in conjunction with Hoechst 33342 to measure cell cycle progression.

    Techniques: TUNEL Assay, FACS, Fluorescence

    The effects of CLK2 on radiation sensitivity of CCD-18Lu cells. Cells were transfected with either vacant vector or myc-CLK2 . At 24 h post-transfection, cells were exposed to 0.05–2 Gy of γ-rays and cell viability was assayed 48 h later. A , the overexpression of Myc-CLK2 at 72 h post-transfection. B , the cell viability of CLK2 overexpressing CCD-18Lu cells to ionizing radiation was determined by MTT assay. C , cell proliferation of CLK2 overexpressing cells after irradiation. D , effect of CLK2 overexpression on 2 Gy-induced apoptosis. DNA fragmentation was detected with HT TiterTACS Assay Kit for the quantification of apoptosis. B–D , data represent mean ± S.D. ( n = 3) and were analyzed by the t test. Data showed a significant difference compared with vector control at the indicated dose (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation of CLK2 at Serine 34 and Threonine 127 by AKT Controls Cell Survival after Ionizing Radiation *

    doi: 10.1074/jbc.M110.122044

    Figure Lengend Snippet: The effects of CLK2 on radiation sensitivity of CCD-18Lu cells. Cells were transfected with either vacant vector or myc-CLK2 . At 24 h post-transfection, cells were exposed to 0.05–2 Gy of γ-rays and cell viability was assayed 48 h later. A , the overexpression of Myc-CLK2 at 72 h post-transfection. B , the cell viability of CLK2 overexpressing CCD-18Lu cells to ionizing radiation was determined by MTT assay. C , cell proliferation of CLK2 overexpressing cells after irradiation. D , effect of CLK2 overexpression on 2 Gy-induced apoptosis. DNA fragmentation was detected with HT TiterTACS Assay Kit for the quantification of apoptosis. B–D , data represent mean ± S.D. ( n = 3) and were analyzed by the t test. Data showed a significant difference compared with vector control at the indicated dose (*, p

    Article Snippet: For the quantification of apoptosis, DNA fragmentation was detected using HT TiterTACS Assay Kit according to the manufacturer's instructions (Trevigen, Inc., Gaithersburg, MD).

    Techniques: Transfection, Plasmid Preparation, Over Expression, MTT Assay, Irradiation

    The regulation of the sensitivity of CCD-18Lu cells to ionizing radiation is dependent on CLK2 phosphorylation. A , CCD-18Lu cells were transfected with vacant vector, WT- CLK2 , or CLK2 mutants and the overexpressions of WT-CLK2 and CLK2 mutants were detected. B , the viabilities of vector, WT-CLK2, or mutant-CLK2 overexpressing cells were measured using MTT assays 48 h after 2 Gy of γ-ray. C , effect of CLK2 phosphorylation on 2 Gy-induced apoptosis. DNA fragmentation was detected with HT TiterTACS Assay Kit for the quantification of apoptosis. The absorbance (450 nm) of untreated vector control was 0.34. D , the viabilities of vacant vector, WT-CLK2, or mutant-CLK2 overexpressing cells after exposure to 0.05 Gy of γ-ray were measured by MTT assay. E , the cell proliferation after irradiation of 0.05 Gy in vacant vector, WT-CLK2, or mutant-CLK2 CCD-18Lu cells. Data represent mean ± S.D. ( n = 3) and were analyzed by Dunnett's test for multiple comparison in analysis of variance to irradiated vector control (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation of CLK2 at Serine 34 and Threonine 127 by AKT Controls Cell Survival after Ionizing Radiation *

    doi: 10.1074/jbc.M110.122044

    Figure Lengend Snippet: The regulation of the sensitivity of CCD-18Lu cells to ionizing radiation is dependent on CLK2 phosphorylation. A , CCD-18Lu cells were transfected with vacant vector, WT- CLK2 , or CLK2 mutants and the overexpressions of WT-CLK2 and CLK2 mutants were detected. B , the viabilities of vector, WT-CLK2, or mutant-CLK2 overexpressing cells were measured using MTT assays 48 h after 2 Gy of γ-ray. C , effect of CLK2 phosphorylation on 2 Gy-induced apoptosis. DNA fragmentation was detected with HT TiterTACS Assay Kit for the quantification of apoptosis. The absorbance (450 nm) of untreated vector control was 0.34. D , the viabilities of vacant vector, WT-CLK2, or mutant-CLK2 overexpressing cells after exposure to 0.05 Gy of γ-ray were measured by MTT assay. E , the cell proliferation after irradiation of 0.05 Gy in vacant vector, WT-CLK2, or mutant-CLK2 CCD-18Lu cells. Data represent mean ± S.D. ( n = 3) and were analyzed by Dunnett's test for multiple comparison in analysis of variance to irradiated vector control (*, p

    Article Snippet: For the quantification of apoptosis, DNA fragmentation was detected using HT TiterTACS Assay Kit according to the manufacturer's instructions (Trevigen, Inc., Gaithersburg, MD).

    Techniques: Transfection, Plasmid Preparation, Mutagenesis, MTT Assay, Irradiation