Structured Review

Promega exonuclease iii
Schema of ReDFISH technique. ( a ) ReDFISH of a chromosome that has replicated fully in the presence of <t>BrdUrd/dC.</t> Newly synthesized DNA incorporating BrdUrd/dC (horizontal stripes) is removed after nicking the DNA with Hoechst 33258 plus UV and digesting nicked DNA with exonuclease <t>III,</t> leaving only the parental strands. The G-rich telomeric strand is the template for lagging strand synthesis and anneals to a Cy3-conjugated C-rich telomeric probe, whereas the C-rich telomeric strand is the template for leading strand synthesis and anneals to an FITC-conjugated G-rich telomeric probe. This pattern defines which telomeric strands replicated at the time of BrdUrd/BrdC labeling. ( b ) ReDFISH of a partially replicated chromosome. In this example, only the p-arm telomere of the X chromosome was replicating during the 1-h BrdUrd/dC pulse; the q arm was not replicating. As a consequence, only the parental strands are available for hybridization in the p arm (schema, p arms), whereas both strands of unreplicated q-arm DNA survive digestion and hybridize to both probes (schema, q arms).
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1) Product Images from "Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes"

Article Title: Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes

Journal:

doi: 10.1073/pnas.0404106101

Schema of ReDFISH technique. ( a ) ReDFISH of a chromosome that has replicated fully in the presence of BrdUrd/dC. Newly synthesized DNA incorporating BrdUrd/dC (horizontal stripes) is removed after nicking the DNA with Hoechst 33258 plus UV and digesting nicked DNA with exonuclease III, leaving only the parental strands. The G-rich telomeric strand is the template for lagging strand synthesis and anneals to a Cy3-conjugated C-rich telomeric probe, whereas the C-rich telomeric strand is the template for leading strand synthesis and anneals to an FITC-conjugated G-rich telomeric probe. This pattern defines which telomeric strands replicated at the time of BrdUrd/BrdC labeling. ( b ) ReDFISH of a partially replicated chromosome. In this example, only the p-arm telomere of the X chromosome was replicating during the 1-h BrdUrd/dC pulse; the q arm was not replicating. As a consequence, only the parental strands are available for hybridization in the p arm (schema, p arms), whereas both strands of unreplicated q-arm DNA survive digestion and hybridize to both probes (schema, q arms).
Figure Legend Snippet: Schema of ReDFISH technique. ( a ) ReDFISH of a chromosome that has replicated fully in the presence of BrdUrd/dC. Newly synthesized DNA incorporating BrdUrd/dC (horizontal stripes) is removed after nicking the DNA with Hoechst 33258 plus UV and digesting nicked DNA with exonuclease III, leaving only the parental strands. The G-rich telomeric strand is the template for lagging strand synthesis and anneals to a Cy3-conjugated C-rich telomeric probe, whereas the C-rich telomeric strand is the template for leading strand synthesis and anneals to an FITC-conjugated G-rich telomeric probe. This pattern defines which telomeric strands replicated at the time of BrdUrd/BrdC labeling. ( b ) ReDFISH of a partially replicated chromosome. In this example, only the p-arm telomere of the X chromosome was replicating during the 1-h BrdUrd/dC pulse; the q arm was not replicating. As a consequence, only the parental strands are available for hybridization in the p arm (schema, p arms), whereas both strands of unreplicated q-arm DNA survive digestion and hybridize to both probes (schema, q arms).

Techniques Used: Synthesized, Labeling, Hybridization

2) Product Images from "PRL-3 promotes telomere deprotection and chromosomal instability"

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx392

Overexpression of PRL-3 promotes telomere dysfunction. ( A ) Validation of PRL-3 stable overexpression. WI38 fibroblasts were infected with control or PRL-3-expressing letivirus. Expression vectors pcDNA3-myc-PRL-3 (for HCT116 cells), pcDNA3.1-myc-PRL-3 (for LoVo cells) and the respective control plasmids were transfected into cells, followed by selection and pooling of stable colonies. Cell lysates were examined by western blot with antibodies to PRL-3, TRF2 and RAP1. ( B ) Effects of PRL-3 stable overexpression on γH2AX, pCHK1 and p53 levels. Indicated cells were treated with ETP (20 μM) or DMSO (1:1000) for 4 h. ( C ) Effects of PRL-3 stable overexpression on TIF formation. WI38 cells were analyzed by IF-FISH staining of pATM (green) and telomere (red). Left, representative staining. Arrows, foci of TIFs. Scale bar, 5 μm. Right, quantification of cells with ≥5 TIFs. Mean ± SD of two independent experiments. n > 60 metaphase per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on dysfunctional telomere repair pathways. Upper, representative CO-FISH staining of WI38 cells. Metaphase cells were stained with probes specific for leading (red) and lagging (green) strands and counterstained with DAPI (blue). Yellow arrow, a typical T-SCE. White arrow, a chromosome–chromosome fusion. Red arrowhead, a MTS. Scale bar, 2.5 μm. Lower, quantification of abnormalities. Mean ± SD of two independent experiments. n > 1300 chromosomes per single experiment. Student's t -test. ( E ) Southern blot analysis of PRL-3 stable overexpression-induced telomere deprotection. Genomic DNA from indicated cells were resolved on agarose gel, transferred to nitrocellulose membrane and probed with biotin-labeled telomere probe. ( F ) qPCR analysis of PRL-3 stable overexpression-induced telomere deprotection. Relative telomere to single copy gene (T/S) ratio of control cells was set as 1. Mean ± SD of three independent experiments. n = 4 replicates per single experiment. Student's t -test.
Figure Legend Snippet: Overexpression of PRL-3 promotes telomere dysfunction. ( A ) Validation of PRL-3 stable overexpression. WI38 fibroblasts were infected with control or PRL-3-expressing letivirus. Expression vectors pcDNA3-myc-PRL-3 (for HCT116 cells), pcDNA3.1-myc-PRL-3 (for LoVo cells) and the respective control plasmids were transfected into cells, followed by selection and pooling of stable colonies. Cell lysates were examined by western blot with antibodies to PRL-3, TRF2 and RAP1. ( B ) Effects of PRL-3 stable overexpression on γH2AX, pCHK1 and p53 levels. Indicated cells were treated with ETP (20 μM) or DMSO (1:1000) for 4 h. ( C ) Effects of PRL-3 stable overexpression on TIF formation. WI38 cells were analyzed by IF-FISH staining of pATM (green) and telomere (red). Left, representative staining. Arrows, foci of TIFs. Scale bar, 5 μm. Right, quantification of cells with ≥5 TIFs. Mean ± SD of two independent experiments. n > 60 metaphase per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on dysfunctional telomere repair pathways. Upper, representative CO-FISH staining of WI38 cells. Metaphase cells were stained with probes specific for leading (red) and lagging (green) strands and counterstained with DAPI (blue). Yellow arrow, a typical T-SCE. White arrow, a chromosome–chromosome fusion. Red arrowhead, a MTS. Scale bar, 2.5 μm. Lower, quantification of abnormalities. Mean ± SD of two independent experiments. n > 1300 chromosomes per single experiment. Student's t -test. ( E ) Southern blot analysis of PRL-3 stable overexpression-induced telomere deprotection. Genomic DNA from indicated cells were resolved on agarose gel, transferred to nitrocellulose membrane and probed with biotin-labeled telomere probe. ( F ) qPCR analysis of PRL-3 stable overexpression-induced telomere deprotection. Relative telomere to single copy gene (T/S) ratio of control cells was set as 1. Mean ± SD of three independent experiments. n = 4 replicates per single experiment. Student's t -test.

Techniques Used: Over Expression, Infection, Expressing, Transfection, Selection, Western Blot, Fluorescence In Situ Hybridization, Staining, Southern Blot, Agarose Gel Electrophoresis, Labeling, Real-time Polymerase Chain Reaction

Overexpression of PRL-3 promotes chromosomal instability and senescence. ( A ) Effects of PRL-3 stable overexpression on APB and MN formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. Student's t -test. n > 1500 cells scored per sample for MN or n > 60 anaphase cells scored per sample for APB. ( B ) Effects of PRL-3 stable overexpression on BrdU incorporation. Indicated cells were treated with double-thymidine block, released into fresh medium containing 10 μM BrdU and incubated for 45 min. Cells were fixed, immunostained with anti-BrdU (green), and counterstained with DAPI (blue). Left, representative staining of BrdU. Scale bar, 15 μm. Right, quantification of BrdU-positive cells. Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on senescence. Indicated cells were treated with DMSO (1:1000) or Ku55933 (5 μM) for 24 h, followed by β-galactosidase staining. Left, representative staining. Right, quantification of β-galactosidase positive cells. Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on H3K9me3 levels. Indicated cells were fixed, immunostained with anti-H3K9me3 (red), and counterstained with DAPI (blue). ( E ) Effects of reconstituted PRL-3 on telomere length, DNA damage and senescence in PRL-3 stable knockdown cells. HCT116 control and PRL-3 stable knockdown cells were co-transfected with indicated amount of pcDNA3 and pcDNA3-PRL-3 plasmids. The total amount of plasmids for each sample was adjusted to 4 μg. After 72 h, protein lysates were subjected to western blot of PRL-3, γH2AX, H3K9me3 (lower). Genomic DNA was used for qPCR analysis of telomere length (upper). Protein bands were scanned and relative OD was calculated by normalizing to GAPDH. T/S ratio of HCT116 control cells transfected with pcDNA3 was set as 1. Pearson χ2 test.
Figure Legend Snippet: Overexpression of PRL-3 promotes chromosomal instability and senescence. ( A ) Effects of PRL-3 stable overexpression on APB and MN formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. Student's t -test. n > 1500 cells scored per sample for MN or n > 60 anaphase cells scored per sample for APB. ( B ) Effects of PRL-3 stable overexpression on BrdU incorporation. Indicated cells were treated with double-thymidine block, released into fresh medium containing 10 μM BrdU and incubated for 45 min. Cells were fixed, immunostained with anti-BrdU (green), and counterstained with DAPI (blue). Left, representative staining of BrdU. Scale bar, 15 μm. Right, quantification of BrdU-positive cells. Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on senescence. Indicated cells were treated with DMSO (1:1000) or Ku55933 (5 μM) for 24 h, followed by β-galactosidase staining. Left, representative staining. Right, quantification of β-galactosidase positive cells. Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on H3K9me3 levels. Indicated cells were fixed, immunostained with anti-H3K9me3 (red), and counterstained with DAPI (blue). ( E ) Effects of reconstituted PRL-3 on telomere length, DNA damage and senescence in PRL-3 stable knockdown cells. HCT116 control and PRL-3 stable knockdown cells were co-transfected with indicated amount of pcDNA3 and pcDNA3-PRL-3 plasmids. The total amount of plasmids for each sample was adjusted to 4 μg. After 72 h, protein lysates were subjected to western blot of PRL-3, γH2AX, H3K9me3 (lower). Genomic DNA was used for qPCR analysis of telomere length (upper). Protein bands were scanned and relative OD was calculated by normalizing to GAPDH. T/S ratio of HCT116 control cells transfected with pcDNA3 was set as 1. Pearson χ2 test.

Techniques Used: Over Expression, Staining, BrdU Incorporation Assay, Blocking Assay, Incubation, Transfection, Western Blot, Real-time Polymerase Chain Reaction

Silencing of PRL-3 promotes DDR and senescence. ( A ) Efficiencies of PRL-3 silencing in HCT116 (knockdown by two shRNAs using lentivirus system, left) and SW480 (knockout by CRISPR/Cas9 system, right) cells and its effects on indicated protein levels. WT, wild-type. KO, knockout. ( B ) Effects of PRL-3 silencing on phosphorylations of H2AX and CHK1. Samples treated with 20 μM etoposide (ETP) for 4 h were used as positive controls. ( C ) Effects of PRL-3 silencing on TIF formation. Indicated HCT116 cells were subjected to IF-FISH staining. Upper, representative staining. Arrows, colocalizations between γH2AX and telomere (TIFs). Scale bar, 5 μm. Lower, quantification of cells with ≥5 TIF. Mean ± SD of two independent experiments. n > 200 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 silencing on anaphase bridges (APB) and micronuclei (MN) formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. n > 1000 cells scored per sample for MN and n > 50 anaphase cells scored per sample for APB. Student's t -test. Representative images of APB (red arrow) and MN (white arrow) of HCT116 cells stained with DAPI were shown. ( E ) ChIP analysis of RAP1 and TRF2's binding to telomeric or Alu DNA in HCT116 and S480 cells silenced for PRL-3. Upper, representative blots after ChIP with indicated antibodies or IgG. Input, 2% DNA. Lower, quantification of relative OD. Relative OD was calculated by normalizing to that of input and relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( F ) PRL-3 silencing induced ROS-dependent cellular senescence and DNA damage response. Indica ted HCT116 cells were treated with NAC (10 mM), GSH (10 mM) or DMSO (1:1000) for 24 h. Part of cells were fixed and processed for β-galactosidase staining, others were analyzed by western blot. Upper, representative β-galactosidase staining of cells treated with DMSO. Middle, quantification of β-galactosidase positive cells. Mean ± SD of two independent experiments. n > 400 cells per single experiment. Student's t -test. Lower, western blot of γH2AX.
Figure Legend Snippet: Silencing of PRL-3 promotes DDR and senescence. ( A ) Efficiencies of PRL-3 silencing in HCT116 (knockdown by two shRNAs using lentivirus system, left) and SW480 (knockout by CRISPR/Cas9 system, right) cells and its effects on indicated protein levels. WT, wild-type. KO, knockout. ( B ) Effects of PRL-3 silencing on phosphorylations of H2AX and CHK1. Samples treated with 20 μM etoposide (ETP) for 4 h were used as positive controls. ( C ) Effects of PRL-3 silencing on TIF formation. Indicated HCT116 cells were subjected to IF-FISH staining. Upper, representative staining. Arrows, colocalizations between γH2AX and telomere (TIFs). Scale bar, 5 μm. Lower, quantification of cells with ≥5 TIF. Mean ± SD of two independent experiments. n > 200 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 silencing on anaphase bridges (APB) and micronuclei (MN) formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. n > 1000 cells scored per sample for MN and n > 50 anaphase cells scored per sample for APB. Student's t -test. Representative images of APB (red arrow) and MN (white arrow) of HCT116 cells stained with DAPI were shown. ( E ) ChIP analysis of RAP1 and TRF2's binding to telomeric or Alu DNA in HCT116 and S480 cells silenced for PRL-3. Upper, representative blots after ChIP with indicated antibodies or IgG. Input, 2% DNA. Lower, quantification of relative OD. Relative OD was calculated by normalizing to that of input and relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( F ) PRL-3 silencing induced ROS-dependent cellular senescence and DNA damage response. Indica ted HCT116 cells were treated with NAC (10 mM), GSH (10 mM) or DMSO (1:1000) for 24 h. Part of cells were fixed and processed for β-galactosidase staining, others were analyzed by western blot. Upper, representative β-galactosidase staining of cells treated with DMSO. Middle, quantification of β-galactosidase positive cells. Mean ± SD of two independent experiments. n > 400 cells per single experiment. Student's t -test. Lower, western blot of γH2AX.

Techniques Used: Knock-Out, CRISPR, Gene Knockout, Fluorescence In Situ Hybridization, Staining, Chromatin Immunoprecipitation, Binding Assay, Western Blot

RAP1 and TRF2-dependent recruitment of PRL-3 to telomere. ( A ) In situ PLA analysis of PRL-3's associations with RAP1 and TRF2. HCT116 cells were pre-extracted, fixed, inmunostained with indicated pairs of antibodies and probed with Duolink in situ PLA reagent. Binding foci were in red and dashed lines indicated outline of nucleus (determined by DAPI counter staining). Scale bar, 10 μm. ( B ) TRF2- and RAP1-dependent recruitment of PRL-3 to telomeric DNA in vitro . Purified myc-TRF2 (150 ng), His-RAP1 (120 ng), and His-PRL-3 (30 ng) were co-incubated with 1 μg biotin-labeled telomere (lanes 1–4) or Alu (lanes 5–8) probe as indicated and subjected to pull-down analysis with Streptavidin agarose. Precipitates were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. ( C and D ) TRF2 and RAP1-dependent recruitment of PRL-3 to telomere in cells. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h, pre-extracted, fixed and subjected to IF-FISH staining. (C) Representative PRL-3 association with telomere. Scale bar, 10 μm. Areas in white squares were enlarged. (D) Quantification of cells with ≥5 associations between PRL-3 foci and telomere. Mean ± SD of three independent experiments. n > 100 cells per single experiment. Student's t -test. ( E ) Knockdown efficiencies of RAP1 and TRF2. HCT116 cells were transfected with 50 nM siRNAs against RAP1 or TRF2 for 48 h. Lysates were analyzed by western blot with indicated antibodies. ( F ) ChIP analysis of PRL-3 binding to telomeric and Alu DNA. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h and processed for ChIP using anti-PRL-3 or pre-immune IgG. Upper, representative blots of hybridization with probe to telomere or Alu. Input, 2% DNA. Lower, quantification of relative optical densities (OD). Relative OD was calculated by normalizing to OD of Input and relative OD of control siRNA-transfected sample was set as 100%. Mean ± SD of three independent experiments. Student's t -test.
Figure Legend Snippet: RAP1 and TRF2-dependent recruitment of PRL-3 to telomere. ( A ) In situ PLA analysis of PRL-3's associations with RAP1 and TRF2. HCT116 cells were pre-extracted, fixed, inmunostained with indicated pairs of antibodies and probed with Duolink in situ PLA reagent. Binding foci were in red and dashed lines indicated outline of nucleus (determined by DAPI counter staining). Scale bar, 10 μm. ( B ) TRF2- and RAP1-dependent recruitment of PRL-3 to telomeric DNA in vitro . Purified myc-TRF2 (150 ng), His-RAP1 (120 ng), and His-PRL-3 (30 ng) were co-incubated with 1 μg biotin-labeled telomere (lanes 1–4) or Alu (lanes 5–8) probe as indicated and subjected to pull-down analysis with Streptavidin agarose. Precipitates were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. ( C and D ) TRF2 and RAP1-dependent recruitment of PRL-3 to telomere in cells. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h, pre-extracted, fixed and subjected to IF-FISH staining. (C) Representative PRL-3 association with telomere. Scale bar, 10 μm. Areas in white squares were enlarged. (D) Quantification of cells with ≥5 associations between PRL-3 foci and telomere. Mean ± SD of three independent experiments. n > 100 cells per single experiment. Student's t -test. ( E ) Knockdown efficiencies of RAP1 and TRF2. HCT116 cells were transfected with 50 nM siRNAs against RAP1 or TRF2 for 48 h. Lysates were analyzed by western blot with indicated antibodies. ( F ) ChIP analysis of PRL-3 binding to telomeric and Alu DNA. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h and processed for ChIP using anti-PRL-3 or pre-immune IgG. Upper, representative blots of hybridization with probe to telomere or Alu. Input, 2% DNA. Lower, quantification of relative optical densities (OD). Relative OD was calculated by normalizing to OD of Input and relative OD of control siRNA-transfected sample was set as 100%. Mean ± SD of three independent experiments. Student's t -test.

Techniques Used: In Situ, Proximity Ligation Assay, Binding Assay, Staining, In Vitro, Purification, Incubation, Labeling, Western Blot, Transfection, Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Hybridization

PRL-3 relocates RAP1 and TRF2 from telomeric DNA. ( A ) Effects of PRL-3 stable overexpression on the chromatin abundance of RAP1, TRF2 and TRF1. Nuclei from HCT116 cells were homogenized in buffer containing indicated concentrations of NaCl. Chromatin-enriched fractions were analyzed by western blot. Left, representative blots. Right, relative levels of TRF2, RAP1 and TRF1. Protein band were scanned and relative OD was calculated by normalizing to OD of H2B. The relative OD of sample prepared with 150 mM NaCl was set as 100%. Mean ± SD of three independent experiments. ANOVA. ( B ) Effects of PRL-3 stable overexpression on bindings of RAP1 and TRF2 to telomeric and Alu DNA. Indicated cells were crosslinked, immunoprecipitated with antibodies to RAP1, TRF2 or pre-immune IgG, and precipitated DNA was analyzed by ChIP. Upper, representative blots. Lower, quantification of relative OD, which was calculated by normalizing to that of Input. Relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on telomere associations of RAP1 and TRF2 in WI38 cells. Left, representative IF-FISH staining of telomere (red) and RAP1 or TRF2 (green). Arrows, foci of co-localization. Scale bar, 10 μm. Right, quantification of cells with ≥5 associations between RAP1 or TRF2 foci and telomere. Mean ± SD of two independent experiments. n > 80 cells per single experiment. Student's t -test. ( D ) EMSA analysis of PRL-3, RAP1 and TRF2's associations with telomeric DNA. Indicated concentrations of purified FLAG-TRF2, His-RAP1, myc-PRL-3 were co-incubated with Biotin-labeled telomere probe (20 nM). To induce super-shift, 0.1 μg anti-PRL-3 (lane 5), anti-TRF2 (lanes 6 and 18) and IgG (lane 7) were used. Note that anti-PRL-3 and anti-TRF2-induced super-shifts of Complex II partially co-migrated with Complex I (lanes 5 and 6).
Figure Legend Snippet: PRL-3 relocates RAP1 and TRF2 from telomeric DNA. ( A ) Effects of PRL-3 stable overexpression on the chromatin abundance of RAP1, TRF2 and TRF1. Nuclei from HCT116 cells were homogenized in buffer containing indicated concentrations of NaCl. Chromatin-enriched fractions were analyzed by western blot. Left, representative blots. Right, relative levels of TRF2, RAP1 and TRF1. Protein band were scanned and relative OD was calculated by normalizing to OD of H2B. The relative OD of sample prepared with 150 mM NaCl was set as 100%. Mean ± SD of three independent experiments. ANOVA. ( B ) Effects of PRL-3 stable overexpression on bindings of RAP1 and TRF2 to telomeric and Alu DNA. Indicated cells were crosslinked, immunoprecipitated with antibodies to RAP1, TRF2 or pre-immune IgG, and precipitated DNA was analyzed by ChIP. Upper, representative blots. Lower, quantification of relative OD, which was calculated by normalizing to that of Input. Relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on telomere associations of RAP1 and TRF2 in WI38 cells. Left, representative IF-FISH staining of telomere (red) and RAP1 or TRF2 (green). Arrows, foci of co-localization. Scale bar, 10 μm. Right, quantification of cells with ≥5 associations between RAP1 or TRF2 foci and telomere. Mean ± SD of two independent experiments. n > 80 cells per single experiment. Student's t -test. ( D ) EMSA analysis of PRL-3, RAP1 and TRF2's associations with telomeric DNA. Indicated concentrations of purified FLAG-TRF2, His-RAP1, myc-PRL-3 were co-incubated with Biotin-labeled telomere probe (20 nM). To induce super-shift, 0.1 μg anti-PRL-3 (lane 5), anti-TRF2 (lanes 6 and 18) and IgG (lane 7) were used. Note that anti-PRL-3 and anti-TRF2-induced super-shifts of Complex II partially co-migrated with Complex I (lanes 5 and 6).

Techniques Used: Over Expression, Western Blot, Immunoprecipitation, Chromatin Immunoprecipitation, Fluorescence In Situ Hybridization, Staining, Purification, Incubation, Labeling

Disrupting PRL-3-RAP1 complex or expressing ectopic TRF2 attenuates PRL-3 overexpression-promoted telomere deprotection, DNA damage, chromosomal instability and senescence. ( A ) HCT116 control and PRL-3 overexpressing cells were transfected with 0.5 μg of pEGFP-N1-Myb or pEGFP-N1 plasmid for 72 h, and indicated proteins were analyzed by western blot. ( B ) qPCR analysis of telomere length of cells in (A). T/S ratio of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test. ( C ) Quantification of micronuclei of cells in (A). Mean ± SD of two independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Quantification of β-galactosidase-positive cells in (A). Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( E ) Relative migration of cells in (A). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of two independent experiments. Three replicates per single experiment. Student's t -test. ( F ) HCT116 control and PRL-3 overexpressing cells were infected with control (Lv-con) or TRF2-expressing lentivirus (Lv-TRF2) for 120 h, and lysates were subjected to western blot. ( G ) qPCR analysis of telomere length of cells in (F). T/S ratio of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. 3 replicates per single experiment. Student's t -test. ( H ) Quantification of micronuclei of cells in (F). Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( I ) Quantification of β-galactosidase-positive cells in (F). Mean ± SD of three independent experiments. n > 300 cells per single experiment. Student's t -test. ( J ) Relative migration of cells of (F). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test.
Figure Legend Snippet: Disrupting PRL-3-RAP1 complex or expressing ectopic TRF2 attenuates PRL-3 overexpression-promoted telomere deprotection, DNA damage, chromosomal instability and senescence. ( A ) HCT116 control and PRL-3 overexpressing cells were transfected with 0.5 μg of pEGFP-N1-Myb or pEGFP-N1 plasmid for 72 h, and indicated proteins were analyzed by western blot. ( B ) qPCR analysis of telomere length of cells in (A). T/S ratio of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test. ( C ) Quantification of micronuclei of cells in (A). Mean ± SD of two independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Quantification of β-galactosidase-positive cells in (A). Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( E ) Relative migration of cells in (A). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of two independent experiments. Three replicates per single experiment. Student's t -test. ( F ) HCT116 control and PRL-3 overexpressing cells were infected with control (Lv-con) or TRF2-expressing lentivirus (Lv-TRF2) for 120 h, and lysates were subjected to western blot. ( G ) qPCR analysis of telomere length of cells in (F). T/S ratio of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. 3 replicates per single experiment. Student's t -test. ( H ) Quantification of micronuclei of cells in (F). Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( I ) Quantification of β-galactosidase-positive cells in (F). Mean ± SD of three independent experiments. n > 300 cells per single experiment. Student's t -test. ( J ) Relative migration of cells of (F). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test.

Techniques Used: Expressing, Over Expression, Transfection, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Migration, Infection

3) Product Images from "Human RECQL1 participates in telomere maintenance"

Article Title: Human RECQL1 participates in telomere maintenance

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku200

RECQL1 associates to telomeres in ALT cells. ( A ) Telomeric ChIP was performed in U2OS cells using anti-RECQL1 antibody in the presence and absence of HU (treated with 5 mM for 18 h). The precipitated DNA was hybridized with the sty-11 telomeric probe (top) or an Alu probe (bottom). Dot blots were performed using anti-RECQL1 antibody, normal IgG and anti-TRF1 antibody that is used as a positive control. ( B ) Telomeric ChIP samples were confirmed by western blotting for the presence of RECQL1 and TRF1. ( C ) The graph showing the signal/input ratio from the RECQL1 IPs as a function of the quantity of chromatin (input). The results were visualized by Phosphor Imager and quantitated with Image Quant software (Molecular Dynamics) and are expressed in % binding to the telomeres. The error bars represent the mean and standard deviation from three independent ChIP assays.
Figure Legend Snippet: RECQL1 associates to telomeres in ALT cells. ( A ) Telomeric ChIP was performed in U2OS cells using anti-RECQL1 antibody in the presence and absence of HU (treated with 5 mM for 18 h). The precipitated DNA was hybridized with the sty-11 telomeric probe (top) or an Alu probe (bottom). Dot blots were performed using anti-RECQL1 antibody, normal IgG and anti-TRF1 antibody that is used as a positive control. ( B ) Telomeric ChIP samples were confirmed by western blotting for the presence of RECQL1 and TRF1. ( C ) The graph showing the signal/input ratio from the RECQL1 IPs as a function of the quantity of chromatin (input). The results were visualized by Phosphor Imager and quantitated with Image Quant software (Molecular Dynamics) and are expressed in % binding to the telomeres. The error bars represent the mean and standard deviation from three independent ChIP assays.

Techniques Used: Chromatin Immunoprecipitation, Positive Control, Western Blot, Software, Binding Assay, Standard Deviation

4) Product Images from "Alternative lengthening of human telomeres is a conservative DNA replication process with features of break‐induced replication"

Article Title: Alternative lengthening of human telomeres is a conservative DNA replication process with features of break‐induced replication

Journal: EMBO Reports

doi: 10.15252/embr.201643169

Conservative DNA replication at telomeres of human ALT cells Examples of chromosome arms exhibiting features of telomeric semiconservative (Semi) replication, conservative (Consrv) replication of the entire telomere (E) and conservative replication of part of the telomere (P). Idealized chromosome diagrams (left) and actual microscopy images (right) are shown. D‐FISH, denaturing FISH; ND‐CO‐FISH, non‐denaturing CO‐FISH. Percentages of chromosome arms exhibiting conservative (Consrv) replication of the entire telomere (E), conservative replication of part of the telomere (P), and conservative replication of either the entire telomere of part of it (E+P). NE, U2OS cells expressing normal levels of cyclin E; OE, U2OS cells overexpressing cyclin E for 4 days. Bars represent means and standard errors of the mean from three independent experiments. Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres: P
Figure Legend Snippet: Conservative DNA replication at telomeres of human ALT cells Examples of chromosome arms exhibiting features of telomeric semiconservative (Semi) replication, conservative (Consrv) replication of the entire telomere (E) and conservative replication of part of the telomere (P). Idealized chromosome diagrams (left) and actual microscopy images (right) are shown. D‐FISH, denaturing FISH; ND‐CO‐FISH, non‐denaturing CO‐FISH. Percentages of chromosome arms exhibiting conservative (Consrv) replication of the entire telomere (E), conservative replication of part of the telomere (P), and conservative replication of either the entire telomere of part of it (E+P). NE, U2OS cells expressing normal levels of cyclin E; OE, U2OS cells overexpressing cyclin E for 4 days. Bars represent means and standard errors of the mean from three independent experiments. Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres: P

Techniques Used: Microscopy, Fluorescence In Situ Hybridization, Expressing, Over Expression

Triple‐FISH protocol to distinguish between telomeric semiconservative and conservative DNA replication Semiconservative replication. Diagram of a chromosome with the telomeric C‐rich and G‐rich strands colored red and green, respectively. Newly synthesized strands are indicated by dotted lines. The three steps of the protocol were strand‐specific: dual color, denaturing FISH (1), non‐denaturing chromosome orientation (CO)‐FISH (2), and denaturing FISH (3). In the second step, the nascent strands have been digested. In all steps, two sets of PNA primers specific for the G‐strand and C‐strand, respectively, were used to monitor the presence of both strands. The arrows indicate the color of the emitted light and its intensity (idealized). Conservative replication. Diagram showing that telomeric conservative replication (shown here to involve the entire length of the telomeres at the p arms) leads to distinct staining patterns, not observed with semiconservative replication. BIR, break‐induced replication. Partially conservative and partially semiconservative telomeric replication. Diagram showing the staining patterns predicted for telomeres that are partially conservatively replicated (distal half) and partially semiconservatively replicated (proximal half), as might occur following fork collapse within a telomere (shown only for the telomeres at the p arms).
Figure Legend Snippet: Triple‐FISH protocol to distinguish between telomeric semiconservative and conservative DNA replication Semiconservative replication. Diagram of a chromosome with the telomeric C‐rich and G‐rich strands colored red and green, respectively. Newly synthesized strands are indicated by dotted lines. The three steps of the protocol were strand‐specific: dual color, denaturing FISH (1), non‐denaturing chromosome orientation (CO)‐FISH (2), and denaturing FISH (3). In the second step, the nascent strands have been digested. In all steps, two sets of PNA primers specific for the G‐strand and C‐strand, respectively, were used to monitor the presence of both strands. The arrows indicate the color of the emitted light and its intensity (idealized). Conservative replication. Diagram showing that telomeric conservative replication (shown here to involve the entire length of the telomeres at the p arms) leads to distinct staining patterns, not observed with semiconservative replication. BIR, break‐induced replication. Partially conservative and partially semiconservative telomeric replication. Diagram showing the staining patterns predicted for telomeres that are partially conservatively replicated (distal half) and partially semiconservatively replicated (proximal half), as might occur following fork collapse within a telomere (shown only for the telomeres at the p arms).

Techniques Used: Fluorescence In Situ Hybridization, Synthesized, Staining

5) Product Images from "Telomere Reprogramming and Maintenance in Porcine iPS Cells"

Article Title: Telomere Reprogramming and Maintenance in Porcine iPS Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074202

DNA damage and telomere dysfunction-induced foci (TIF) in porcine iPS cells. (A, C, E and G) Percentage of γH2Ax positive cells. Cells are categorized into three groups with fewer than 10, 10–30 and more than 30 γH2Ax foci, respectively. n = number of cells counted. *p
Figure Legend Snippet: DNA damage and telomere dysfunction-induced foci (TIF) in porcine iPS cells. (A, C, E and G) Percentage of γH2Ax positive cells. Cells are categorized into three groups with fewer than 10, 10–30 and more than 30 γH2Ax foci, respectively. n = number of cells counted. *p

Techniques Used:

6) Product Images from "cis and trans Requirements for Rolling Circle Replication of a Satellite RNA"

Article Title: cis and trans Requirements for Rolling Circle Replication of a Satellite RNA

Journal:

doi: 10.1128/JVI.78.6.3072-3082.2004

Secondary structure of satRPV RNA monomer. (A) Autoradiograph of 5′-end-labeled transcript of monomeric satRPV RNA after partial digestion with imidazole or RNase T1 . Gel-purified, end-labeled RNA was incubated in three different molarities of imidazole under nondenaturing (native) conditions for 15 h (lanes 3 to 6). Indicated units of RNase T1 were used for digestion in nondenaturing (native) conditions for 5 min at 25°C. To generate the G-track sequencing ladder, indicated units of RNase T1 were incubated with RNA under denaturing conditions for 5 min at 50°C (left two lanes). The positions of G residues are indicated at left, numbered from the 5′ end. The positions of L1 and L2a sequences and nucleotides 60 to 76 are indicated at right. (B) Secondary-structure model of satRPV RNA predicted by MFOLD superimposed with markers indicating intensity of cleavages in panel A. Open, filled, and double symbols indicate weak, moderate, and strong cuts, respectively.
Figure Legend Snippet: Secondary structure of satRPV RNA monomer. (A) Autoradiograph of 5′-end-labeled transcript of monomeric satRPV RNA after partial digestion with imidazole or RNase T1 . Gel-purified, end-labeled RNA was incubated in three different molarities of imidazole under nondenaturing (native) conditions for 15 h (lanes 3 to 6). Indicated units of RNase T1 were used for digestion in nondenaturing (native) conditions for 5 min at 25°C. To generate the G-track sequencing ladder, indicated units of RNase T1 were incubated with RNA under denaturing conditions for 5 min at 50°C (left two lanes). The positions of G residues are indicated at left, numbered from the 5′ end. The positions of L1 and L2a sequences and nucleotides 60 to 76 are indicated at right. (B) Secondary-structure model of satRPV RNA predicted by MFOLD superimposed with markers indicating intensity of cleavages in panel A. Open, filled, and double symbols indicate weak, moderate, and strong cuts, respectively.

Techniques Used: Autoradiography, Labeling, Purification, Incubation, Sequencing

7) Product Images from "Functional interaction between DNA-PKcs and telomerase in telomere length maintenance"

Article Title: Functional interaction between DNA-PKcs and telomerase in telomere length maintenance

Journal:

doi: 10.1093/emboj/cdf593

Fig. 2. Telomere length determination using Q-FISH on testis sections. ( A ) Average telomere fluorescence in arbitrary units (a.u.f.) of 100 meiotic cells from each mouse of the indicated genotype. Standard deviation, as well as the total number of meiocytes analyzed per mouse are indicated. ( B ) Average telomere fluorescence in arbitrary units (a.u.f.) of three or four mice grouped per genotype. Standard deviation, as well as the total number of mice of each genotype, are indicated. There was a significant decrease in average telomere fluorescence in G1 Terc–/– /DNA-PKcs–/– meiotic cells compared with G1 Terc–/– /DNA-PKcs+/+ controls ( P < 0.001).
Figure Legend Snippet: Fig. 2. Telomere length determination using Q-FISH on testis sections. ( A ) Average telomere fluorescence in arbitrary units (a.u.f.) of 100 meiotic cells from each mouse of the indicated genotype. Standard deviation, as well as the total number of meiocytes analyzed per mouse are indicated. ( B ) Average telomere fluorescence in arbitrary units (a.u.f.) of three or four mice grouped per genotype. Standard deviation, as well as the total number of mice of each genotype, are indicated. There was a significant decrease in average telomere fluorescence in G1 Terc–/– /DNA-PKcs–/– meiotic cells compared with G1 Terc–/– /DNA-PKcs+/+ controls ( P < 0.001).

Techniques Used: Fluorescence In Situ Hybridization, Fluorescence, Standard Deviation, Mouse Assay

8) Product Images from "Telomeric RNAs are essential to maintain telomeres"

Article Title: Telomeric RNAs are essential to maintain telomeres

Journal: Nature Communications

doi: 10.1038/ncomms12534

Deletion of the 20q-TERRA locus decreases telomere length and protection in U2OS cells. ( a ) Q-FISH images obtained from metaphases spreads from U2OS cells WT and KO for the Chr20q-TERRA locus (clones A4, B4 and C4). (Left graphs) Frequency graphs of telomere length (a.u.) distribution measured in WT and in the 20q-KO cells (clones A4, B4 and C4) from three independent experiments. The mean telomere length and the number of telomeres and metaphases analyzed is shown. The red lines are arbitrary lines placed in the exact same position in each frequency graph to visualize differences between the 20q-KO clones and the WT controls (right graphs) The mean telomere length, the percentage of short telomeres and the quantification of signal-free ends per metaphase are also represented. Short telomeres are considered those in the 10% percentile of the total telomere length distribution. Total number of metaphases used for the statistical analysis is indicated. Scale bar, 10 μm and (zoom) 1 μm. ( b ) WT and 20q-KO cells were analyzed for T-SCE events with G-rich (green) and C-rich (red) PNA probes. The fraction of chromosome ends with T-SCE obtained from three different experiments was quantified and graphed as the mean values±s.e.m., n =30 metaphases. The number of metaphases analyzed is shown. Only events in which interchange of both colours were quantified (see examples of no-T-SCE and T-SCE). The quantification was carried out by counting the number of events in the same chromosome or in different chromosomes and then normalizing it by the total number of chromosomes observed in each metaphase. Scale bar, 1 μm. ( c ) Quantification of DNA-containing double minute chromosomes (TDMs) in WT and 20q-KO cells from three different experiments (mean values±s.e.m., n =30 metaphases). An example of TDMs is shown. One-way Anova with Dunnett's post test was used for all statistical analysis (* P
Figure Legend Snippet: Deletion of the 20q-TERRA locus decreases telomere length and protection in U2OS cells. ( a ) Q-FISH images obtained from metaphases spreads from U2OS cells WT and KO for the Chr20q-TERRA locus (clones A4, B4 and C4). (Left graphs) Frequency graphs of telomere length (a.u.) distribution measured in WT and in the 20q-KO cells (clones A4, B4 and C4) from three independent experiments. The mean telomere length and the number of telomeres and metaphases analyzed is shown. The red lines are arbitrary lines placed in the exact same position in each frequency graph to visualize differences between the 20q-KO clones and the WT controls (right graphs) The mean telomere length, the percentage of short telomeres and the quantification of signal-free ends per metaphase are also represented. Short telomeres are considered those in the 10% percentile of the total telomere length distribution. Total number of metaphases used for the statistical analysis is indicated. Scale bar, 10 μm and (zoom) 1 μm. ( b ) WT and 20q-KO cells were analyzed for T-SCE events with G-rich (green) and C-rich (red) PNA probes. The fraction of chromosome ends with T-SCE obtained from three different experiments was quantified and graphed as the mean values±s.e.m., n =30 metaphases. The number of metaphases analyzed is shown. Only events in which interchange of both colours were quantified (see examples of no-T-SCE and T-SCE). The quantification was carried out by counting the number of events in the same chromosome or in different chromosomes and then normalizing it by the total number of chromosomes observed in each metaphase. Scale bar, 1 μm. ( c ) Quantification of DNA-containing double minute chromosomes (TDMs) in WT and 20q-KO cells from three different experiments (mean values±s.e.m., n =30 metaphases). An example of TDMs is shown. One-way Anova with Dunnett's post test was used for all statistical analysis (* P

Techniques Used: Fluorescence In Situ Hybridization, Gene Knockout, Clone Assay

Deletion of the 20q-TERRA locus decreases telomere protection in U2OS cells. ( a ) Quantification of the total γH2AX signal per nucleus (mean values±s.e.m., n =number of cells) is shown. The total number of cells analyzed is indicated. ( b ) Quantification of the total 53BP1 spot signal per nucleus (mean values±s.e.m., n =number of cells is shown). The total number of cells analyzed is indicated. ( c ) Graphs showing the quantification of the co-localization (TIF) between TRF2 and either γH2AX or 53BP1 in WT cells and in all 20q-KO clones (mean values±s.e.m., n =3 independent experiments for γH2AX and n =number of cells for 53BP1) per cell is shown. The total number of nuclei analyzed is indicated. ( d ) Representative images of the average number of TIFs found on double inmunostain to detect the telomere protein TRF2 (green) and either the DNA damage markers phospho-Histone γH2AX or 53BP1 (red) in the U2OS cells WT or deleted for the 20q locus. Arrowheads indicate co-localization events. Scale bar, 10 μm. ( e ) Quantification of chromosomal end-to-end fusions in WT and in the 20q-KO cells from three independent experiments (mean values±s.e.m., n =metaphases). Examples of end-to-end fusions are shown as well. Scale bar, 1 μm. ( f ) Array-CGH analysis was performed on hybridization on the same membrane of DNA differentially labelled from WT and 20q-KO cells. The chromosomal gains and losses in 20q-KO cells normalized by WT cells are represented. The chromosomal gains are shown in green and in red the chromosomal losses. One-way Anova with Dunnett's post test was used for all statistical analysis except for the quantification of chromosomal fusions in which the Student's t -test was used (* P
Figure Legend Snippet: Deletion of the 20q-TERRA locus decreases telomere protection in U2OS cells. ( a ) Quantification of the total γH2AX signal per nucleus (mean values±s.e.m., n =number of cells) is shown. The total number of cells analyzed is indicated. ( b ) Quantification of the total 53BP1 spot signal per nucleus (mean values±s.e.m., n =number of cells is shown). The total number of cells analyzed is indicated. ( c ) Graphs showing the quantification of the co-localization (TIF) between TRF2 and either γH2AX or 53BP1 in WT cells and in all 20q-KO clones (mean values±s.e.m., n =3 independent experiments for γH2AX and n =number of cells for 53BP1) per cell is shown. The total number of nuclei analyzed is indicated. ( d ) Representative images of the average number of TIFs found on double inmunostain to detect the telomere protein TRF2 (green) and either the DNA damage markers phospho-Histone γH2AX or 53BP1 (red) in the U2OS cells WT or deleted for the 20q locus. Arrowheads indicate co-localization events. Scale bar, 10 μm. ( e ) Quantification of chromosomal end-to-end fusions in WT and in the 20q-KO cells from three independent experiments (mean values±s.e.m., n =metaphases). Examples of end-to-end fusions are shown as well. Scale bar, 1 μm. ( f ) Array-CGH analysis was performed on hybridization on the same membrane of DNA differentially labelled from WT and 20q-KO cells. The chromosomal gains and losses in 20q-KO cells normalized by WT cells are represented. The chromosomal gains are shown in green and in red the chromosomal losses. One-way Anova with Dunnett's post test was used for all statistical analysis except for the quantification of chromosomal fusions in which the Student's t -test was used (* P

Techniques Used: Gene Knockout, Clone Assay, Hybridization

Related Articles

Clone Assay:

Article Title: Sequencing of long stretches of repetitive DNA
Article Snippet: Aliquots of 5 μg were treated with 500 U of exonuclease III (Promega) at 37 °C for different lengths of time. .. Aliquots of 5 μg were treated with 500 U of exonuclease III (Promega) at 37 °C for different lengths of time.

DNA Synthesis:

Article Title: Functional interaction between DNA-PKcs and telomerase in telomere length maintenance
Article Snippet: Slides were then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymic digestion of the BrdU-substituted DNA strands with 3 U/µl exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM DTT pH 8.0) was allowed to proceed for 10 min at room temperature. .. Slides were then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymic digestion of the BrdU-substituted DNA strands with 3 U/µl exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM DTT pH 8.0) was allowed to proceed for 10 min at room temperature.

Autoradiography:

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus
Article Snippet: Some of the ligation mixtures were digested after termination of ligations with ∼25 units of Exonuclease III (Promega) at 37°C for 30 min. Before electrophoresis, all DNA samples were deproteinized as described in the DNA supercoiling assay. .. Some of the ligation mixtures were digested after termination of ligations with ∼25 units of Exonuclease III (Promega) at 37°C for 30 min. Before electrophoresis, all DNA samples were deproteinized as described in the DNA supercoiling assay.

Construct:

Article Title: cis and trans Requirements for Rolling Circle Replication of a Satellite RNA
Article Snippet: After the second-round inoculation (48 hpi), total RNA was extracted from protoplasts and selected negative-strand mutants were cloned by reverse transcription-PCR with SuperScript II (Invitrogen). .. Serial deletion mutants (see Fig. ) of satRPV were generated by using exonuclease III (ExoIII; Erase-a-Base system; Promega) after digestion of plasmid pWT with Msc I (construct M122), Ava I (A222, A241, A243) or Cla I (C321). .. Deletion mutants were constructed as follows.

Electrophoresis:

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus
Article Snippet: The DNA was then ligated with T4 DNA ligase (0.6 unit/reaction; Promega) at 30°C for 30 min, and the ligation reactions were terminated by incubation of samples at 65°C for 15 min. .. Some of the ligation mixtures were digested after termination of ligations with ∼25 units of Exonuclease III (Promega) at 37°C for 30 min. Before electrophoresis, all DNA samples were deproteinized as described in the DNA supercoiling assay. .. The protein-free DNAs were loaded on pre-run 6% polyacrylamide gels in 0.5× TBE buffer, and finally resolved at 200 V for 3 h at 4°C.

Incubation:

Article Title: Alternative lengthening of human telomeres is a conservative DNA replication process with features of break‐induced replication
Article Snippet: Chromosome preparations were stained with Hoechst 33258 (0.5 μg/ml) (Sigma), incubated in 2× SSC (Invitrogen) for 15 min at RT, and exposed to 365‐nm UV light (Stratalinker 1800 UV irradiator) for 45 min. .. The 5′‐bromo‐2′‐deoxyuridine‐ and 5′‐bromo‐2′‐deoxycytosine‐substituted DNA was digested with exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 , and 50 mM Tris–HCl, pH 8.0) for 15 min at 37°C.

Article Title: Human RECQL1 participates in telomere maintenance
Article Snippet: Cells were treated with 5-bromo-2-deoxyuridine (BrdU) and 5-bromo-2-deoxycytidine (BrdC) (Sigma) (3:1) at a final concentration of 1 × 10−5 m for 16 h, followed by incubation with 0.1 mg/ml colcemid (Invitrogen) for 4 h. The cells were then suspended in prewarmed 75 mM KCl hypotonic solution for 20 min at 37ºC followed by 3:1 methanol/acetic acid fixation. .. Slides were then rinsed with 2× SSC and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 30 min, and then digested the DNA with 10 U/µl exonuclease III (Promega) for 10 min at RT.

Article Title: Functional interaction between DNA-PKcs and telomerase in telomere length maintenance
Article Snippet: Briefly, confluent cell monolayers were subcultured into medium containing 5′-bromo-2′-deoxyuridine (BrdU; Sigma, St Louis, MO) at a total final concentration of 1 × 10–5 M then incubated at 37°C for an additional 24 h. Colcemid (0.2 µg/ml) was added during the final 4 h to accumulate mitotic cells. .. Slides were then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymic digestion of the BrdU-substituted DNA strands with 3 U/µl exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM DTT pH 8.0) was allowed to proceed for 10 min at room temperature.

Article Title: Telomere Reprogramming and Maintenance in Porcine iPS Cells
Article Snippet: Chromosome preparations were treated with 0.5 mg/ml RNase-A (Roche) for 10 min at 37°C, stained with Hoechst 33258 (0.5 μg/ml) (Sigma), incubated in 2xSSC (Invitrogen) for 15 min at room temperature (RT) and exposed to 365-nm UV light (Stratalinker 1800 UV irradiator) for 30 min. .. The 5′-bromo-2′-deoxyuridine-substituted DNA was digested with Exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 and 50 mM Tris-HCl, pH 8.0) for 10 min at RT.

Article Title: MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end-resection
Article Snippet: In brief, for CO-FISH cells were grown in 10 μM BrdU:BrdC (3:1) for 24 h with the addition of 0.2 μg/ml demecolcine for the final 2 h. Slides were treated with RNAse A (0.5 mg/ml) for 10 min at 37 °C, stained with Hoechst 33258 (0.5 μg/ml) in 2xSSC for 15 min at room temperature and exposed to 5.4e3 J/m2 365-nm UV light (Stratalinker 2400 UV irradiator). .. Following digestion with Exonuclease III (10 U/μl, Promega, Madison, WI) for 10 min at room temperature, slides were dehydrated through an ethanol series (70%, 95% and 100%) and incubated sequentially with Cy3-TelG 5′-[TTAGGG]3 -3′ and FITC-TelC 5′-[CCCTAA]3 -3′ probes at room temperature. .. Telomere mobility analysis was done as described with minor adjustments.

Article Title: Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes
Article Snippet: The nicked BrdUrd/dC-substituted DNA was digested with 3 units/μl exonuclease III (Promega) in 50 mM Tris·HCl (pH 8.0), 5 mM MgCl2 , and 5 mM DTT for 10 min at room temperature. .. The C-rich strands (templates for leading strand synthesis) were revealed by hybridizing (20 μl) in 70% formamide, 20 ng of Cy3-conjugated (TTAGGG)3 2′-deoxyoligonucleotide N3′ -P5′ phosphoramidate probe ( , ), 0.25% (wt/vol) blocking reagent (Roche), and 5% MgCl2 in 10 mM Tris, pH 7.2, which was added to the slides containing single-stranded chromosomal target DNA.

Article Title: A Distinct Class of Genome Rearrangements Driven by Heterologous Recombination
Article Snippet: Trypsinized cells were incubated in 75 mM KCl and fixed with methanol:acetic acid (3:1 ratio). .. The BrdU-labeled DNA strands were digested with 100 μl Exonuclease III (Promega) at 10 U/ μl, in the buffer supplied by the manufacturer, for 30 min at room temperature.

Article Title: A role for monoubiquitinated FANCD2 at telomeres in ALT cells
Article Snippet: Briefly, U2OS or HeLa cells were grown overnight and then incubated with 10 µM bromodeoxyuridine (BrdU) for 16 h, with 1 µg/ml nocodazole added for the last 5 h. Cells were collected following trypsinization and were resuspended in 75 mM KCl for 20 min prior to fixation and preparation of metaphase spreads. .. Following incubation with Hoechst 33258, treatment with UV light (using a Stratalinker 1800 UV irradiator), and digestion with Exonuclease III (Promega), cells were hybridized overnight with 0.5 ng of Cy3-(TTAGGG)3 telomeric probe. .. Slides were then washed and mounted with a coverslip and Vectashield containing DAPI.

Article Title: Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis
Article Snippet: Following irradiation, cell cultures were incubated for various times, trypsinized, and subcultured into medium containing 5-bromo-2-deoxyuridine (BrdU, 10 μM; Sigma-Aldrich) for one cell cycle. .. Following UV exposure, BrdU incorporated strands were digested with Exonuclease III (3 U/μL in provided reaction buffer; Promega) at room temperature for 10 min.

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus
Article Snippet: The DNA was then ligated with T4 DNA ligase (0.6 unit/reaction; Promega) at 30°C for 30 min, and the ligation reactions were terminated by incubation of samples at 65°C for 15 min. .. Some of the ligation mixtures were digested after termination of ligations with ∼25 units of Exonuclease III (Promega) at 37°C for 30 min. Before electrophoresis, all DNA samples were deproteinized as described in the DNA supercoiling assay.

Article Title: The helicase domain of Polθ counteracts RPA to promote alt-NHEJ
Article Snippet: The next day, the slides were rehydrated with PBS for 5 min, treated with 0.5 mg/ml RNase A (in PBS, DNase free) for 10 min at 37°C, incubated with 0.5 ∝g/ml Hoechst 33258 (Sigma) in 2XSSC for 15 min at room temperature, and exposed to 365-nm UV light (Stratalinker 1800 UV irradiator) for 30 min. .. The slides were then digested twice with 800 U Exonuclease III (Promega) at room temperature for 10 min each, washed with PBS and dehydrated through an ethanol series of 70%, 95%, 100%.

Expressing:

Article Title: The helicase domain of Polθ counteracts RPA to promote alt-NHEJ
Article Snippet: 6 hours post-infection, cells were treated with 4-OHT to induce the expression of CRE and subsequent loss of shelterin. .. The slides were then digested twice with 800 U Exonuclease III (Promega) at room temperature for 10 min each, washed with PBS and dehydrated through an ethanol series of 70%, 95%, 100%.

Modification:

Article Title: Functional interaction between DNA-PKcs and telomerase in telomere length maintenance
Article Snippet: CO-FISH has been described in detail previously and was used here with some modification ( ). .. Slides were then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymic digestion of the BrdU-substituted DNA strands with 3 U/µl exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM DTT pH 8.0) was allowed to proceed for 10 min at room temperature.

Article Title: A role for monoubiquitinated FANCD2 at telomeres in ALT cells
Article Snippet: T-SCE was analyzed by chromosome orientation fluorescence in situ hybridization (CO-FISH) as previously described , with slight modification. .. Following incubation with Hoechst 33258, treatment with UV light (using a Stratalinker 1800 UV irradiator), and digestion with Exonuclease III (Promega), cells were hybridized overnight with 0.5 ng of Cy3-(TTAGGG)3 telomeric probe.

Article Title: Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis
Article Snippet: Chromosome-orientation fluorescence in situ hybridization (CO-FISH) was employed to evaluate IR-induced chromosomal instability and performed as previously described ( , ) with some modification. .. Following UV exposure, BrdU incorporated strands were digested with Exonuclease III (3 U/μL in provided reaction buffer; Promega) at room temperature for 10 min.

Blocking Assay:

Article Title: Alternative lengthening of human telomeres is a conservative DNA replication process with features of break‐induced replication
Article Snippet: The 5′‐bromo‐2′‐deoxyuridine‐ and 5′‐bromo‐2′‐deoxycytosine‐substituted DNA was digested with exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 , and 50 mM Tris–HCl, pH 8.0) for 15 min at 37°C. .. The 5′‐bromo‐2′‐deoxyuridine‐ and 5′‐bromo‐2′‐deoxycytosine‐substituted DNA was digested with exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 , and 50 mM Tris–HCl, pH 8.0) for 15 min at 37°C.

Article Title: Human RECQL1 participates in telomere maintenance
Article Snippet: Slides were then rinsed with 2× SSC and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 30 min, and then digested the DNA with 10 U/µl exonuclease III (Promega) for 10 min at RT. .. Slides were then rinsed with 2× SSC and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 30 min, and then digested the DNA with 10 U/µl exonuclease III (Promega) for 10 min at RT.

Article Title: Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes
Article Snippet: The nicked BrdUrd/dC-substituted DNA was digested with 3 units/μl exonuclease III (Promega) in 50 mM Tris·HCl (pH 8.0), 5 mM MgCl2 , and 5 mM DTT for 10 min at room temperature. .. The nicked BrdUrd/dC-substituted DNA was digested with 3 units/μl exonuclease III (Promega) in 50 mM Tris·HCl (pH 8.0), 5 mM MgCl2 , and 5 mM DTT for 10 min at room temperature.

Article Title: A Distinct Class of Genome Rearrangements Driven by Heterologous Recombination
Article Snippet: The BrdU-labeled DNA strands were digested with 100 μl Exonuclease III (Promega) at 10 U/ μl, in the buffer supplied by the manufacturer, for 30 min at room temperature. .. The BrdU-labeled DNA strands were digested with 100 μl Exonuclease III (Promega) at 10 U/ μl, in the buffer supplied by the manufacturer, for 30 min at room temperature.

Hybridization:

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability
Article Snippet: Slides were heated at 85°C for 10 min, stained with 50 nM pre-heated (65°C) Cy3-PNA probe (Panagene, Daejeon, Korea) in hybridization buffer (20 mM Na2 HPO4 pH7.4, 20 mM Tris–HCl pH 7.4, 60% formamide, 2× SSC, 0.1 μg/ml salmon sperm DNA) at 85°C for 10 min, then at room temperature for 16 h. Next day, slides were washed with washing buffer I (10 mM Tris–HCl pH 7.2, 70% formamide, 0.1% BSA) for two times, washing buffer II (10 mM Tris–HCl pH 7.2, 1.5 M NaCl, 0.1% Tween 20) for three times, counterstained with 4΄,6-Diamidino-2-phenylindole (DAPI) in 2× SSC and mounted with 50% glycerol. .. The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

Article Title: Alternative lengthening of human telomeres is a conservative DNA replication process with features of break‐induced replication
Article Snippet: The 5′‐bromo‐2′‐deoxyuridine‐ and 5′‐bromo‐2′‐deoxycytosine‐substituted DNA was digested with exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 , and 50 mM Tris–HCl, pH 8.0) for 15 min at 37°C. .. The 5′‐bromo‐2′‐deoxyuridine‐ and 5′‐bromo‐2′‐deoxycytosine‐substituted DNA was digested with exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 , and 50 mM Tris–HCl, pH 8.0) for 15 min at 37°C.

Article Title: Telomeric RNAs are essential to maintain telomeres
Article Snippet: The slides were treated with 0.5 mg ml−1 RNase A for 10 min at 37 °C, stained with 0.5 μg ml−1 Hoechst 33258 (Sigma) in 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate) for 15 min at room temperature and then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymatic digestion of the BrdU/BrdC-substituted DNA strands with 3 U μl−1 of Exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM dithiothreitol, pH 8) was allowed to proceed for 10 min at room temperature. .. The slides were treated with 0.5 mg ml−1 RNase A for 10 min at 37 °C, stained with 0.5 μg ml−1 Hoechst 33258 (Sigma) in 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate) for 15 min at room temperature and then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymatic digestion of the BrdU/BrdC-substituted DNA strands with 3 U μl−1 of Exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM dithiothreitol, pH 8) was allowed to proceed for 10 min at room temperature.

Article Title: Human RECQL1 participates in telomere maintenance
Article Snippet: Slides were then rinsed with 2× SSC and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 30 min, and then digested the DNA with 10 U/µl exonuclease III (Promega) for 10 min at RT. .. Slides were then rinsed with 2× SSC and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 30 min, and then digested the DNA with 10 U/µl exonuclease III (Promega) for 10 min at RT.

Article Title: Functional interaction between DNA-PKcs and telomerase in telomere length maintenance
Article Snippet: Prior to hybridization of the single-stranded (TTAGGG)7 telomere probe, slides were treated with 0.5 mg/ml RNase A for 10 min at 37°C, then stained with 0.5 µg/ml Hoechst 33258 (Sigma) in 2× SSC for 15 min at room temperature. .. Slides were then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymic digestion of the BrdU-substituted DNA strands with 3 U/µl exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM DTT pH 8.0) was allowed to proceed for 10 min at room temperature.

Article Title: Telomere Reprogramming and Maintenance in Porcine iPS Cells
Article Snippet: Paragraph title: Chromatid Orientation Fluorescence In Situ Hybridization (CO-FISH) ... The 5′-bromo-2′-deoxyuridine-substituted DNA was digested with Exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 and 50 mM Tris-HCl, pH 8.0) for 10 min at RT.

Article Title: A Distinct Class of Genome Rearrangements Driven by Heterologous Recombination
Article Snippet: The BrdU-labeled DNA strands were digested with 100 μl Exonuclease III (Promega) at 10 U/ μl, in the buffer supplied by the manufacturer, for 30 min at room temperature. .. The BrdU-labeled DNA strands were digested with 100 μl Exonuclease III (Promega) at 10 U/ μl, in the buffer supplied by the manufacturer, for 30 min at room temperature.

Article Title: Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork
Article Snippet: Fixed chromosomes were UV-sensitized in 0.5 μg/ml Hoechst 33258 (Sigma) in 2× SSC at room temperature for 15 min and exposed to 365 nm UV light for 1 h using a UV cross-linker (Vilber-Lourmat, Marne-la-Vallée, France). .. Exposed chromosomes were digested with 3 units/μl exonuclease III (Promega, Madison, WI) at room temperature for 15 min, denatured in 70% formamide in 2× SSC, and dehydrated in cold ethanol before hybridization. .. Chromosomes were hybridized first using a 0.03 μg/ml concentration of a leading strand telomere PNA probe (FAM-(TTAGGG)3 ) followed by a 0.03 μg/ml concentration of a lagging strand PNA probe (Cy3-(CCCTAA)3 ) (both probes from PNA Bio, Thousand Oaks, CA).

Article Title: Identification of telomere dysfunction in Friedreich ataxia
Article Snippet: Chromosome orientation fluorescence in situ hybridisation (CO-FISH) was performed as previously described [ ]. .. The nicked BrdU-substituted DNA strands were then digested by 3U/ml of Exonuclease III (Promega) in the buffer supplied by the manufacturer at room temperature for 10 min.

Article Title: A role for monoubiquitinated FANCD2 at telomeres in ALT cells
Article Snippet: T-SCE was analyzed by chromosome orientation fluorescence in situ hybridization (CO-FISH) as previously described , with slight modification. .. Following incubation with Hoechst 33258, treatment with UV light (using a Stratalinker 1800 UV irradiator), and digestion with Exonuclease III (Promega), cells were hybridized overnight with 0.5 ng of Cy3-(TTAGGG)3 telomeric probe.

Article Title: The helicase domain of Polθ counteracts RPA to promote alt-NHEJ
Article Snippet: The slides were then digested twice with 800 U Exonuclease III (Promega) at room temperature for 10 min each, washed with PBS and dehydrated through an ethanol series of 70%, 95%, 100%. .. After air-drying, slides were hybridized with Tamra-OO-[TTAGGG]3 PNA probe in hybridization solution (70% formamide, 1 mg/ml blocking reagent (Roche), 10 mM Tris-HCl pH 7.5) for 2 hours at room temperature.

Ligation:

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus
Article Snippet: The DNA was then ligated with T4 DNA ligase (0.6 unit/reaction; Promega) at 30°C for 30 min, and the ligation reactions were terminated by incubation of samples at 65°C for 15 min. .. Some of the ligation mixtures were digested after termination of ligations with ∼25 units of Exonuclease III (Promega) at 37°C for 30 min. Before electrophoresis, all DNA samples were deproteinized as described in the DNA supercoiling assay. .. The protein-free DNAs were loaded on pre-run 6% polyacrylamide gels in 0.5× TBE buffer, and finally resolved at 200 V for 3 h at 4°C.

Generated:

Article Title: cis and trans Requirements for Rolling Circle Replication of a Satellite RNA
Article Snippet: After the second-round inoculation (48 hpi), total RNA was extracted from protoplasts and selected negative-strand mutants were cloned by reverse transcription-PCR with SuperScript II (Invitrogen). .. Serial deletion mutants (see Fig. ) of satRPV were generated by using exonuclease III (ExoIII; Erase-a-Base system; Promega) after digestion of plasmid pWT with Msc I (construct M122), Ava I (A222, A241, A243) or Cla I (C321). .. Deletion mutants were constructed as follows.

Imaging:

Article Title: Identification of telomere dysfunction in Friedreich ataxia
Article Snippet: The nicked BrdU-substituted DNA strands were then digested by 3U/ml of Exonuclease III (Promega) in the buffer supplied by the manufacturer at room temperature for 10 min. .. In situ hybridisation was performed using the Cy3-conjugated PNA telomeric oligonucleotide (CCCTAA)3 probe as previously reported [ ].

Sequencing:

Article Title: Sequencing of long stretches of repetitive DNA
Article Snippet: Paragraph title: Exonuclease digestion of subclones and sequencing ... Aliquots of 5 μg were treated with 500 U of exonuclease III (Promega) at 37 °C for different lengths of time.

Antiviral Assay:

Article Title: cis and trans Requirements for Rolling Circle Replication of a Satellite RNA
Article Snippet: After the second-round inoculation (48 hpi), total RNA was extracted from protoplasts and selected negative-strand mutants were cloned by reverse transcription-PCR with SuperScript II (Invitrogen). .. Serial deletion mutants (see Fig. ) of satRPV were generated by using exonuclease III (ExoIII; Erase-a-Base system; Promega) after digestion of plasmid pWT with Msc I (construct M122), Ava I (A222, A241, A243) or Cla I (C321). .. Deletion mutants were constructed as follows.

Recombinant:

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus
Article Snippet: Briefly, a 32 P-labeled 123-bp DNA fragment (∼1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (25–50 nM) or total protein extracts from adult mosquitos (4 µg) in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µL. .. Some of the ligation mixtures were digested after termination of ligations with ∼25 units of Exonuclease III (Promega) at 37°C for 30 min. Before electrophoresis, all DNA samples were deproteinized as described in the DNA supercoiling assay.

Immunofluorescence:

Article Title: Identification of telomere dysfunction in Friedreich ataxia
Article Snippet: The nicked BrdU-substituted DNA strands were then digested by 3U/ml of Exonuclease III (Promega) in the buffer supplied by the manufacturer at room temperature for 10 min. .. The nicked BrdU-substituted DNA strands were then digested by 3U/ml of Exonuclease III (Promega) in the buffer supplied by the manufacturer at room temperature for 10 min.

Fluorescence:

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability
Article Snippet: Paragraph title: Cytogenetic analysis and fluorescence in situ hybridization (FISH) staining ... The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

Article Title: Telomere Reprogramming and Maintenance in Porcine iPS Cells
Article Snippet: Paragraph title: Chromatid Orientation Fluorescence In Situ Hybridization (CO-FISH) ... The 5′-bromo-2′-deoxyuridine-substituted DNA was digested with Exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 and 50 mM Tris-HCl, pH 8.0) for 10 min at RT.

Article Title: Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes
Article Snippet: One clone of mouse NIH 3T3 cells was mixed with testing samples and used as an internal control to normalize results and to convert the fluorescence intensity measurements of human telomeres to kilobases ( , ). .. The nicked BrdUrd/dC-substituted DNA was digested with 3 units/μl exonuclease III (Promega) in 50 mM Tris·HCl (pH 8.0), 5 mM MgCl2 , and 5 mM DTT for 10 min at room temperature.

Article Title: Identification of telomere dysfunction in Friedreich ataxia
Article Snippet: Chromosome orientation fluorescence in situ hybridisation (CO-FISH) was performed as previously described [ ]. .. The nicked BrdU-substituted DNA strands were then digested by 3U/ml of Exonuclease III (Promega) in the buffer supplied by the manufacturer at room temperature for 10 min.

Article Title: A role for monoubiquitinated FANCD2 at telomeres in ALT cells
Article Snippet: T-SCE was analyzed by chromosome orientation fluorescence in situ hybridization (CO-FISH) as previously described , with slight modification. .. Following incubation with Hoechst 33258, treatment with UV light (using a Stratalinker 1800 UV irradiator), and digestion with Exonuclease III (Promega), cells were hybridized overnight with 0.5 ng of Cy3-(TTAGGG)3 telomeric probe.

Article Title: Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis
Article Snippet: Paragraph title: Chromosome-Orientation Fluorescence In situ Hybridization ... Following UV exposure, BrdU incorporated strands were digested with Exonuclease III (3 U/μL in provided reaction buffer; Promega) at room temperature for 10 min.

Mutagenesis:

Article Title: cis and trans Requirements for Rolling Circle Replication of a Satellite RNA
Article Snippet: Serial deletion mutants (see Fig. ) of satRPV were generated by using exonuclease III (ExoIII; Erase-a-Base system; Promega) after digestion of plasmid pWT with Msc I (construct M122), Ava I (A222, A241, A243) or Cla I (C321). .. Serial deletion mutants (see Fig. ) of satRPV were generated by using exonuclease III (ExoIII; Erase-a-Base system; Promega) after digestion of plasmid pWT with Msc I (construct M122), Ava I (A222, A241, A243) or Cla I (C321).

Microscopy:

Article Title: Functional interaction between DNA-PKcs and telomerase in telomere length maintenance
Article Snippet: Cells, a majority of which are now singly substituted with BrdU, were harvested and metaphase spreads prepared on microscope slides as described above. .. Slides were then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymic digestion of the BrdU-substituted DNA strands with 3 U/µl exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM DTT pH 8.0) was allowed to proceed for 10 min at room temperature.

Article Title: Telomere Reprogramming and Maintenance in Porcine iPS Cells
Article Snippet: The 5′-bromo-2′-deoxyuridine-substituted DNA was digested with Exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 and 50 mM Tris-HCl, pH 8.0) for 10 min at RT. .. PNA-telomere strand specific FISH was performed using heat denaturated Fluorescein-OO-(CCCTAA)3 and TAMRA (6-carboxytetramethylrhodamine)-OO-(TTAGGG)3 (Bio-Synthesis Inc, TX, USA) telomeric probes.

Article Title: Identification of telomere dysfunction in Friedreich ataxia
Article Snippet: Images of interphase cells were acquired on a digital fluorescence microscope (Zeiss Axioskop 2) equipped with a CCD camera (Photometrics) and Smart Capture software (Digital Scientific) using fixed exposure time of 0.5 second and magnification of 63×. .. The nicked BrdU-substituted DNA strands were then digested by 3U/ml of Exonuclease III (Promega) in the buffer supplied by the manufacturer at room temperature for 10 min.

Article Title: Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis
Article Snippet: Following UV exposure, BrdU incorporated strands were digested with Exonuclease III (3 U/μL in provided reaction buffer; Promega) at room temperature for 10 min. .. Following UV exposure, BrdU incorporated strands were digested with Exonuclease III (3 U/μL in provided reaction buffer; Promega) at room temperature for 10 min.

Purification:

Article Title: Sequencing of long stretches of repetitive DNA
Article Snippet: The digested DNA was then purified using the DNA Clean & Concentrator-5 kit (Zymo Research). .. Aliquots of 5 μg were treated with 500 U of exonuclease III (Promega) at 37 °C for different lengths of time.

Polymerase Chain Reaction:

Article Title: Sequencing of long stretches of repetitive DNA
Article Snippet: Aliquots of 5 μg were treated with 500 U of exonuclease III (Promega) at 37 °C for different lengths of time. .. Aliquots of 5 μg were treated with 500 U of exonuclease III (Promega) at 37 °C for different lengths of time.

Labeling:

Article Title: The helicase domain of Polθ counteracts RPA to promote alt-NHEJ
Article Snippet: For the CO-FISH assay, ~50% confluent MEFs were labeled with BrdU:BrdC (3:1, final concentration: 10 ∝M) for 14–16 hours and then incubated for 2 hours with 0.2 ∝g/ml colcemid (Sigma). .. The slides were then digested twice with 800 U Exonuclease III (Promega) at room temperature for 10 min each, washed with PBS and dehydrated through an ethanol series of 70%, 95%, 100%.

Polyacrylamide Gel Electrophoresis:

Article Title: Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages
Article Snippet: 5′-32 P-labeled G*-2S-30, G*-2S-51, C*-2S-32 and C*-2S-51 (1 pmol, 0.025 pmol/µl) and the corresponding template, C-51-Temp or G-51-Temp (1.5 pmol) were mixed with 4 µl of 10 × exonuclease III buffer (final concentration 66 mM Tris–HCl, pH 8.0, 0.66 mM MgCl2 ); final volume adjusted to 40 µl with water. .. The solution was heated at 95°C for 5 min and allowed to cool to room temperature over 1 h. Exonuclease III (Promega, 2 units – 100 units, final concentration 0.05 U/µl – 2.5 U/µl) was added at 22°C and aliquots (5 µl) were taken from each reaction at the appropriate time (2, 5, 15 and 30 min), quenched by 2 µl of 0.5 M EDTA and then mixed with PAGE loading buffer (5 µl, formamide). .. Samples were resolved by electrophoresis using a 20% PAGE (7 M urea).

In Situ Hybridization:

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability
Article Snippet: Paragraph title: Cytogenetic analysis and fluorescence in situ hybridization (FISH) staining ... The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

Article Title: Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis
Article Snippet: Paragraph title: Chromosome-Orientation Fluorescence In situ Hybridization ... Following UV exposure, BrdU incorporated strands were digested with Exonuclease III (3 U/μL in provided reaction buffer; Promega) at room temperature for 10 min.

Plasmid Preparation:

Article Title: Human RECQL1 participates in telomere maintenance
Article Snippet: Slides were then rinsed with 2× SSC and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 30 min, and then digested the DNA with 10 U/µl exonuclease III (Promega) for 10 min at RT. .. Slides were then rinsed with 2× SSC and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 30 min, and then digested the DNA with 10 U/µl exonuclease III (Promega) for 10 min at RT.

Article Title: cis and trans Requirements for Rolling Circle Replication of a Satellite RNA
Article Snippet: After the second-round inoculation (48 hpi), total RNA was extracted from protoplasts and selected negative-strand mutants were cloned by reverse transcription-PCR with SuperScript II (Invitrogen). .. Serial deletion mutants (see Fig. ) of satRPV were generated by using exonuclease III (ExoIII; Erase-a-Base system; Promega) after digestion of plasmid pWT with Msc I (construct M122), Ava I (A222, A241, A243) or Cla I (C321). .. Deletion mutants were constructed as follows.

Article Title: Zscan4 regulates telomere elongation and genomic stability in ES cells
Article Snippet: The BrdU-substituted DNA was digested with 3 units/µl Exonuclease III (Promega) for 10 min at room temperature. .. The leading strand telomeres were revealed by 3'-Cy3-conjugated (TTAGGG)7 (IDA) without denaturation step and incubated overnight at 37°C.

Article Title: Sequencing of long stretches of repetitive DNA
Article Snippet: Aliquots of 5 μg were treated with 500 U of exonuclease III (Promega) at 37 °C for different lengths of time. .. Aliquots of 5 μg were treated with 500 U of exonuclease III (Promega) at 37 °C for different lengths of time.

Article Title: Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis
Article Snippet: Following UV exposure, BrdU incorporated strands were digested with Exonuclease III (3 U/μL in provided reaction buffer; Promega) at room temperature for 10 min. .. Slides were hybridized with a Cy-3 conjugated (TTAGGG)3 PNA telomere probe (0.2 μg/mL; Applied Biosystems) at 37°C for 1.5 h, rinsed in 70% formamide at 32°C for 10 min, and dehydrated in another ethanol series before re-probing at 37°C for 2 h. Following the second hybridization, slides were rinsed with 70% formamide at 32°C for 15 min followed by 5 min rinse in PN buffer.

Software:

Article Title: Telomere Reprogramming and Maintenance in Porcine iPS Cells
Article Snippet: The 5′-bromo-2′-deoxyuridine-substituted DNA was digested with Exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 and 50 mM Tris-HCl, pH 8.0) for 10 min at RT. .. PNA-telomere strand specific FISH was performed using heat denaturated Fluorescein-OO-(CCCTAA)3 and TAMRA (6-carboxytetramethylrhodamine)-OO-(TTAGGG)3 (Bio-Synthesis Inc, TX, USA) telomeric probes.

Article Title: Identification of telomere dysfunction in Friedreich ataxia
Article Snippet: Telomere fluorescence intensity per cell was analysed using IP Lab software (Digital Scientific) and the average signal was evaluated by subtracting the background signal from the total telomeric signal intensity. .. The nicked BrdU-substituted DNA strands were then digested by 3U/ml of Exonuclease III (Promega) in the buffer supplied by the manufacturer at room temperature for 10 min.

Article Title: Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages
Article Snippet: The solution was heated at 95°C for 5 min and allowed to cool to room temperature over 1 h. Exonuclease III (Promega, 2 units – 100 units, final concentration 0.05 U/µl – 2.5 U/µl) was added at 22°C and aliquots (5 µl) were taken from each reaction at the appropriate time (2, 5, 15 and 30 min), quenched by 2 µl of 0.5 M EDTA and then mixed with PAGE loading buffer (5 µl, formamide). .. The solution was heated at 95°C for 5 min and allowed to cool to room temperature over 1 h. Exonuclease III (Promega, 2 units – 100 units, final concentration 0.05 U/µl – 2.5 U/µl) was added at 22°C and aliquots (5 µl) were taken from each reaction at the appropriate time (2, 5, 15 and 30 min), quenched by 2 µl of 0.5 M EDTA and then mixed with PAGE loading buffer (5 µl, formamide).

Article Title: Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis
Article Snippet: Following UV exposure, BrdU incorporated strands were digested with Exonuclease III (3 U/μL in provided reaction buffer; Promega) at room temperature for 10 min. .. Following UV exposure, BrdU incorporated strands were digested with Exonuclease III (3 U/μL in provided reaction buffer; Promega) at room temperature for 10 min.

Irradiation:

Article Title: Alternative lengthening of human telomeres is a conservative DNA replication process with features of break‐induced replication
Article Snippet: Chromosome preparations were stained with Hoechst 33258 (0.5 μg/ml) (Sigma), incubated in 2× SSC (Invitrogen) for 15 min at RT, and exposed to 365‐nm UV light (Stratalinker 1800 UV irradiator) for 45 min. .. The 5′‐bromo‐2′‐deoxyuridine‐ and 5′‐bromo‐2′‐deoxycytosine‐substituted DNA was digested with exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 , and 50 mM Tris–HCl, pH 8.0) for 15 min at 37°C.

Article Title: Telomeric RNAs are essential to maintain telomeres
Article Snippet: Exponentially growing cells were sub-cultured in the presence of 5′-bromo-2′-deoxyuridine (BrdU; Sigma) at a final concentration of 1 × 10−5 M, and then allowed to replicate their DNA once at 37 °C for 24 h. Colcemide (Gibco) was added at a concentration of 0.1 μg ml−1 during the last 4 h. Cells were then recovered and metaphases prepared (see Q-FISH methods). .. The slides were treated with 0.5 mg ml−1 RNase A for 10 min at 37 °C, stained with 0.5 μg ml−1 Hoechst 33258 (Sigma) in 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate) for 15 min at room temperature and then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymatic digestion of the BrdU/BrdC-substituted DNA strands with 3 U μl−1 of Exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM dithiothreitol, pH 8) was allowed to proceed for 10 min at room temperature. .. An additional denaturation in 70% formamide, 2 × SSC at 70 °C for 1 min was performed in order to ensure complete removal of the newly replicated bromo-substituted strands and followed by dehydration in a cold ethanol series (70, 85, 100%).

Article Title: Human RECQL1 participates in telomere maintenance
Article Snippet: The slides were rehydrated in PBS for 15 min followed by 0.5 mg/ml RNAase treatment at 37ºC for 10 min and subsequent 0.5 µg/ml Hoechst 33258 (Sigma) staining in 2× SSC for 15 min at room temperature (RT). .. Slides were then rinsed with 2× SSC and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 30 min, and then digested the DNA with 10 U/µl exonuclease III (Promega) for 10 min at RT. .. Slides were then washed with PBS and series of ethanol dehydration processes, 70%, 90% and 100%, 5 min each.

Article Title: Functional interaction between DNA-PKcs and telomerase in telomere length maintenance
Article Snippet: Prior to hybridization of the single-stranded (TTAGGG)7 telomere probe, slides were treated with 0.5 mg/ml RNase A for 10 min at 37°C, then stained with 0.5 µg/ml Hoechst 33258 (Sigma) in 2× SSC for 15 min at room temperature. .. Slides were then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymic digestion of the BrdU-substituted DNA strands with 3 U/µl exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM DTT pH 8.0) was allowed to proceed for 10 min at room temperature. .. An additional denaturation in 70% formamide, 2× SSC at 70°C for 1 min was performed, followed by dehydration in a cold ethanol series (70, 85 and 100%).

Article Title: Telomere Reprogramming and Maintenance in Porcine iPS Cells
Article Snippet: Chromosome preparations were treated with 0.5 mg/ml RNase-A (Roche) for 10 min at 37°C, stained with Hoechst 33258 (0.5 μg/ml) (Sigma), incubated in 2xSSC (Invitrogen) for 15 min at room temperature (RT) and exposed to 365-nm UV light (Stratalinker 1800 UV irradiator) for 30 min. .. The 5′-bromo-2′-deoxyuridine-substituted DNA was digested with Exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 and 50 mM Tris-HCl, pH 8.0) for 10 min at RT.

Article Title: MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end-resection
Article Snippet: In brief, for CO-FISH cells were grown in 10 μM BrdU:BrdC (3:1) for 24 h with the addition of 0.2 μg/ml demecolcine for the final 2 h. Slides were treated with RNAse A (0.5 mg/ml) for 10 min at 37 °C, stained with Hoechst 33258 (0.5 μg/ml) in 2xSSC for 15 min at room temperature and exposed to 5.4e3 J/m2 365-nm UV light (Stratalinker 2400 UV irradiator). .. Following digestion with Exonuclease III (10 U/μl, Promega, Madison, WI) for 10 min at room temperature, slides were dehydrated through an ethanol series (70%, 95% and 100%) and incubated sequentially with Cy3-TelG 5′-[TTAGGG]3 -3′ and FITC-TelC 5′-[CCCTAA]3 -3′ probes at room temperature.

Article Title: Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes
Article Snippet: Slides from cells grown in the presence of BrdUrd/dC were treated with 0.5 mg/ml ribonuclease A (Roche) for 10 min at 37°C, stained with 0.5 μg/ml Hoechst 33258 (Sigma) for 15 min at room temperature, mounted with McIlvaine's buffer at pH 8.0, and exposed to 365-nm UV light (Stratalinker 1800 UV irradiator) for 30 min at 55°C. .. The nicked BrdUrd/dC-substituted DNA was digested with 3 units/μl exonuclease III (Promega) in 50 mM Tris·HCl (pH 8.0), 5 mM MgCl2 , and 5 mM DTT for 10 min at room temperature.

Article Title: Zscan4 regulates telomere elongation and genomic stability in ES cells
Article Snippet: Slides were stained with 0.5 µg/ml Hoechst 33258 (Sigma), washed in 2× SSC for 20 min at room temperature, mounted with McIlvaine’s buffer (at pH 8.0), and exposed to 365-nm UV light (Stratalinker 1800 UV irradiator) for 30 min. .. The BrdU-substituted DNA was digested with 3 units/µl Exonuclease III (Promega) for 10 min at room temperature.

Article Title: A Distinct Class of Genome Rearrangements Driven by Heterologous Recombination
Article Snippet: The slides were rehydrated in PBS for 5 minutes, treated with 0.5 mg/ml RNase A (in 1X PBS) for 15 min at 37°C, stained with 0.5 μg/ml Hoechst 33258 (in 2X SSC) for 20 min at room temperature and finally irradiated at 365 nm UV light (Stratalinker 1800 UV, 5.4 × 103 J/m2 ) for 45 min. .. The BrdU-labeled DNA strands were digested with 100 μl Exonuclease III (Promega) at 10 U/ μl, in the buffer supplied by the manufacturer, for 30 min at room temperature.

Article Title: Identification of telomere dysfunction in Friedreich ataxia
Article Snippet: Briefly, cells were grown in the presence of BrdU/BrdC (3:1) (Sigma Aldrich) at a concentration of 1 × 10−5 for 24 h. Slides containing chromosome preparation were stained with DNA-binding fluorescent dye Hoechst 33258 (0.5 μg/ml; Sigma Aldrich) for 15 min at room temperature and were exposed to 365 nm UV light (Stratelinker 1800 UV irradiator) for 30 min. .. The nicked BrdU-substituted DNA strands were then digested by 3U/ml of Exonuclease III (Promega) in the buffer supplied by the manufacturer at room temperature for 10 min.

Article Title: A role for monoubiquitinated FANCD2 at telomeres in ALT cells
Article Snippet: Briefly, U2OS or HeLa cells were grown overnight and then incubated with 10 µM bromodeoxyuridine (BrdU) for 16 h, with 1 µg/ml nocodazole added for the last 5 h. Cells were collected following trypsinization and were resuspended in 75 mM KCl for 20 min prior to fixation and preparation of metaphase spreads. .. Following incubation with Hoechst 33258, treatment with UV light (using a Stratalinker 1800 UV irradiator), and digestion with Exonuclease III (Promega), cells were hybridized overnight with 0.5 ng of Cy3-(TTAGGG)3 telomeric probe. .. Slides were then washed and mounted with a coverslip and Vectashield containing DAPI.

Article Title: Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis
Article Snippet: Following irradiation, cell cultures were incubated for various times, trypsinized, and subcultured into medium containing 5-bromo-2-deoxyuridine (BrdU, 10 μM; Sigma-Aldrich) for one cell cycle. .. Following UV exposure, BrdU incorporated strands were digested with Exonuclease III (3 U/μL in provided reaction buffer; Promega) at room temperature for 10 min.

Article Title: The helicase domain of Polθ counteracts RPA to promote alt-NHEJ
Article Snippet: The next day, the slides were rehydrated with PBS for 5 min, treated with 0.5 mg/ml RNase A (in PBS, DNase free) for 10 min at 37°C, incubated with 0.5 ∝g/ml Hoechst 33258 (Sigma) in 2XSSC for 15 min at room temperature, and exposed to 365-nm UV light (Stratalinker 1800 UV irradiator) for 30 min. .. The slides were then digested twice with 800 U Exonuclease III (Promega) at room temperature for 10 min each, washed with PBS and dehydrated through an ethanol series of 70%, 95%, 100%.

In Vitro:

Article Title: cis and trans Requirements for Rolling Circle Replication of a Satellite RNA
Article Snippet: Paragraph title: Generation of deletion mutants for in vitro transcription. ... Serial deletion mutants (see Fig. ) of satRPV were generated by using exonuclease III (ExoIII; Erase-a-Base system; Promega) after digestion of plasmid pWT with Msc I (construct M122), Ava I (A222, A241, A243) or Cla I (C321).

BrdU Incorporation Assay:

Article Title: Zscan4 regulates telomere elongation and genomic stability in ES cells
Article Snippet: On the 3rd day 5'-bromo-2'-deoxyuridine (BrdU) was added for 12 hours to allow BrdU incorporation for one cell cycle. .. The BrdU-substituted DNA was digested with 3 units/µl Exonuclease III (Promega) for 10 min at room temperature.

Produced:

Article Title: Functional interaction between DNA-PKcs and telomerase in telomere length maintenance
Article Snippet: Slides were then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymic digestion of the BrdU-substituted DNA strands with 3 U/µl exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM DTT pH 8.0) was allowed to proceed for 10 min at room temperature. .. Slides were then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymic digestion of the BrdU-substituted DNA strands with 3 U/µl exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM DTT pH 8.0) was allowed to proceed for 10 min at room temperature.

Concentration Assay:

Article Title: Telomeric RNAs are essential to maintain telomeres
Article Snippet: Exponentially growing cells were sub-cultured in the presence of 5′-bromo-2′-deoxyuridine (BrdU; Sigma) at a final concentration of 1 × 10−5 M, and then allowed to replicate their DNA once at 37 °C for 24 h. Colcemide (Gibco) was added at a concentration of 0.1 μg ml−1 during the last 4 h. Cells were then recovered and metaphases prepared (see Q-FISH methods). .. The slides were treated with 0.5 mg ml−1 RNase A for 10 min at 37 °C, stained with 0.5 μg ml−1 Hoechst 33258 (Sigma) in 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate) for 15 min at room temperature and then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymatic digestion of the BrdU/BrdC-substituted DNA strands with 3 U μl−1 of Exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM dithiothreitol, pH 8) was allowed to proceed for 10 min at room temperature.

Article Title: Human RECQL1 participates in telomere maintenance
Article Snippet: Cells were treated with 5-bromo-2-deoxyuridine (BrdU) and 5-bromo-2-deoxycytidine (BrdC) (Sigma) (3:1) at a final concentration of 1 × 10−5 m for 16 h, followed by incubation with 0.1 mg/ml colcemid (Invitrogen) for 4 h. The cells were then suspended in prewarmed 75 mM KCl hypotonic solution for 20 min at 37ºC followed by 3:1 methanol/acetic acid fixation. .. Slides were then rinsed with 2× SSC and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 30 min, and then digested the DNA with 10 U/µl exonuclease III (Promega) for 10 min at RT.

Article Title: Functional interaction between DNA-PKcs and telomerase in telomere length maintenance
Article Snippet: Briefly, confluent cell monolayers were subcultured into medium containing 5′-bromo-2′-deoxyuridine (BrdU; Sigma, St Louis, MO) at a total final concentration of 1 × 10–5 M then incubated at 37°C for an additional 24 h. Colcemid (0.2 µg/ml) was added during the final 4 h to accumulate mitotic cells. .. Slides were then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymic digestion of the BrdU-substituted DNA strands with 3 U/µl exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM DTT pH 8.0) was allowed to proceed for 10 min at room temperature.

Article Title: Identification of telomere dysfunction in Friedreich ataxia
Article Snippet: Briefly, cells were grown in the presence of BrdU/BrdC (3:1) (Sigma Aldrich) at a concentration of 1 × 10−5 for 24 h. Slides containing chromosome preparation were stained with DNA-binding fluorescent dye Hoechst 33258 (0.5 μg/ml; Sigma Aldrich) for 15 min at room temperature and were exposed to 365 nm UV light (Stratelinker 1800 UV irradiator) for 30 min. .. The nicked BrdU-substituted DNA strands were then digested by 3U/ml of Exonuclease III (Promega) in the buffer supplied by the manufacturer at room temperature for 10 min.

Article Title: Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages
Article Snippet: 5′-32 P-labeled G*-2S-30, G*-2S-51, C*-2S-32 and C*-2S-51 (1 pmol, 0.025 pmol/µl) and the corresponding template, C-51-Temp or G-51-Temp (1.5 pmol) were mixed with 4 µl of 10 × exonuclease III buffer (final concentration 66 mM Tris–HCl, pH 8.0, 0.66 mM MgCl2 ); final volume adjusted to 40 µl with water. .. The solution was heated at 95°C for 5 min and allowed to cool to room temperature over 1 h. Exonuclease III (Promega, 2 units – 100 units, final concentration 0.05 U/µl – 2.5 U/µl) was added at 22°C and aliquots (5 µl) were taken from each reaction at the appropriate time (2, 5, 15 and 30 min), quenched by 2 µl of 0.5 M EDTA and then mixed with PAGE loading buffer (5 µl, formamide). .. Samples were resolved by electrophoresis using a 20% PAGE (7 M urea).

Article Title: The helicase domain of Polθ counteracts RPA to promote alt-NHEJ
Article Snippet: For the CO-FISH assay, ~50% confluent MEFs were labeled with BrdU:BrdC (3:1, final concentration: 10 ∝M) for 14–16 hours and then incubated for 2 hours with 0.2 ∝g/ml colcemid (Sigma). .. The slides were then digested twice with 800 U Exonuclease III (Promega) at room temperature for 10 min each, washed with PBS and dehydrated through an ethanol series of 70%, 95%, 100%.

In Situ:

Article Title: Telomere Reprogramming and Maintenance in Porcine iPS Cells
Article Snippet: Paragraph title: Chromatid Orientation Fluorescence In Situ Hybridization (CO-FISH) ... The 5′-bromo-2′-deoxyuridine-substituted DNA was digested with Exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 and 50 mM Tris-HCl, pH 8.0) for 10 min at RT.

Article Title: Identification of telomere dysfunction in Friedreich ataxia
Article Snippet: Chromosome orientation fluorescence in situ hybridisation (CO-FISH) was performed as previously described [ ]. .. The nicked BrdU-substituted DNA strands were then digested by 3U/ml of Exonuclease III (Promega) in the buffer supplied by the manufacturer at room temperature for 10 min.

Article Title: A role for monoubiquitinated FANCD2 at telomeres in ALT cells
Article Snippet: T-SCE was analyzed by chromosome orientation fluorescence in situ hybridization (CO-FISH) as previously described , with slight modification. .. Following incubation with Hoechst 33258, treatment with UV light (using a Stratalinker 1800 UV irradiator), and digestion with Exonuclease III (Promega), cells were hybridized overnight with 0.5 ng of Cy3-(TTAGGG)3 telomeric probe.

Staining:

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability
Article Snippet: Paragraph title: Cytogenetic analysis and fluorescence in situ hybridization (FISH) staining ... The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

Article Title: Alternative lengthening of human telomeres is a conservative DNA replication process with features of break‐induced replication
Article Snippet: Chromosome preparations were stained with Hoechst 33258 (0.5 μg/ml) (Sigma), incubated in 2× SSC (Invitrogen) for 15 min at RT, and exposed to 365‐nm UV light (Stratalinker 1800 UV irradiator) for 45 min. .. The 5′‐bromo‐2′‐deoxyuridine‐ and 5′‐bromo‐2′‐deoxycytosine‐substituted DNA was digested with exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 , and 50 mM Tris–HCl, pH 8.0) for 15 min at 37°C.

Article Title: Telomeric RNAs are essential to maintain telomeres
Article Snippet: Exponentially growing cells were sub-cultured in the presence of 5′-bromo-2′-deoxyuridine (BrdU; Sigma) at a final concentration of 1 × 10−5 M, and then allowed to replicate their DNA once at 37 °C for 24 h. Colcemide (Gibco) was added at a concentration of 0.1 μg ml−1 during the last 4 h. Cells were then recovered and metaphases prepared (see Q-FISH methods). .. The slides were treated with 0.5 mg ml−1 RNase A for 10 min at 37 °C, stained with 0.5 μg ml−1 Hoechst 33258 (Sigma) in 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate) for 15 min at room temperature and then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymatic digestion of the BrdU/BrdC-substituted DNA strands with 3 U μl−1 of Exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM dithiothreitol, pH 8) was allowed to proceed for 10 min at room temperature. .. An additional denaturation in 70% formamide, 2 × SSC at 70 °C for 1 min was performed in order to ensure complete removal of the newly replicated bromo-substituted strands and followed by dehydration in a cold ethanol series (70, 85, 100%).

Article Title: Human RECQL1 participates in telomere maintenance
Article Snippet: The slides were rehydrated in PBS for 15 min followed by 0.5 mg/ml RNAase treatment at 37ºC for 10 min and subsequent 0.5 µg/ml Hoechst 33258 (Sigma) staining in 2× SSC for 15 min at room temperature (RT). .. Slides were then rinsed with 2× SSC and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 30 min, and then digested the DNA with 10 U/µl exonuclease III (Promega) for 10 min at RT.

Article Title: Functional interaction between DNA-PKcs and telomerase in telomere length maintenance
Article Snippet: Prior to hybridization of the single-stranded (TTAGGG)7 telomere probe, slides were treated with 0.5 mg/ml RNase A for 10 min at 37°C, then stained with 0.5 µg/ml Hoechst 33258 (Sigma) in 2× SSC for 15 min at room temperature. .. Slides were then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymic digestion of the BrdU-substituted DNA strands with 3 U/µl exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM DTT pH 8.0) was allowed to proceed for 10 min at room temperature.

Article Title: Telomere Reprogramming and Maintenance in Porcine iPS Cells
Article Snippet: Chromosome preparations were treated with 0.5 mg/ml RNase-A (Roche) for 10 min at 37°C, stained with Hoechst 33258 (0.5 μg/ml) (Sigma), incubated in 2xSSC (Invitrogen) for 15 min at room temperature (RT) and exposed to 365-nm UV light (Stratalinker 1800 UV irradiator) for 30 min. .. The 5′-bromo-2′-deoxyuridine-substituted DNA was digested with Exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 and 50 mM Tris-HCl, pH 8.0) for 10 min at RT.

Article Title: MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end-resection
Article Snippet: In brief, for CO-FISH cells were grown in 10 μM BrdU:BrdC (3:1) for 24 h with the addition of 0.2 μg/ml demecolcine for the final 2 h. Slides were treated with RNAse A (0.5 mg/ml) for 10 min at 37 °C, stained with Hoechst 33258 (0.5 μg/ml) in 2xSSC for 15 min at room temperature and exposed to 5.4e3 J/m2 365-nm UV light (Stratalinker 2400 UV irradiator). .. Following digestion with Exonuclease III (10 U/μl, Promega, Madison, WI) for 10 min at room temperature, slides were dehydrated through an ethanol series (70%, 95% and 100%) and incubated sequentially with Cy3-TelG 5′-[TTAGGG]3 -3′ and FITC-TelC 5′-[CCCTAA]3 -3′ probes at room temperature.

Article Title: Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes
Article Snippet: Slides from cells grown in the presence of BrdUrd/dC were treated with 0.5 mg/ml ribonuclease A (Roche) for 10 min at 37°C, stained with 0.5 μg/ml Hoechst 33258 (Sigma) for 15 min at room temperature, mounted with McIlvaine's buffer at pH 8.0, and exposed to 365-nm UV light (Stratalinker 1800 UV irradiator) for 30 min at 55°C. .. The nicked BrdUrd/dC-substituted DNA was digested with 3 units/μl exonuclease III (Promega) in 50 mM Tris·HCl (pH 8.0), 5 mM MgCl2 , and 5 mM DTT for 10 min at room temperature.

Article Title: Zscan4 regulates telomere elongation and genomic stability in ES cells
Article Snippet: Slides were stained with 0.5 µg/ml Hoechst 33258 (Sigma), washed in 2× SSC for 20 min at room temperature, mounted with McIlvaine’s buffer (at pH 8.0), and exposed to 365-nm UV light (Stratalinker 1800 UV irradiator) for 30 min. .. The BrdU-substituted DNA was digested with 3 units/µl Exonuclease III (Promega) for 10 min at room temperature.

Article Title: A Distinct Class of Genome Rearrangements Driven by Heterologous Recombination
Article Snippet: The slides were rehydrated in PBS for 5 minutes, treated with 0.5 mg/ml RNase A (in 1X PBS) for 15 min at 37°C, stained with 0.5 μg/ml Hoechst 33258 (in 2X SSC) for 20 min at room temperature and finally irradiated at 365 nm UV light (Stratalinker 1800 UV, 5.4 × 103 J/m2 ) for 45 min. .. The BrdU-labeled DNA strands were digested with 100 μl Exonuclease III (Promega) at 10 U/ μl, in the buffer supplied by the manufacturer, for 30 min at room temperature.

Article Title: Identification of telomere dysfunction in Friedreich ataxia
Article Snippet: Briefly, cells were grown in the presence of BrdU/BrdC (3:1) (Sigma Aldrich) at a concentration of 1 × 10−5 for 24 h. Slides containing chromosome preparation were stained with DNA-binding fluorescent dye Hoechst 33258 (0.5 μg/ml; Sigma Aldrich) for 15 min at room temperature and were exposed to 365 nm UV light (Stratelinker 1800 UV irradiator) for 30 min. .. The nicked BrdU-substituted DNA strands were then digested by 3U/ml of Exonuclease III (Promega) in the buffer supplied by the manufacturer at room temperature for 10 min.

Article Title: A preclinical mouse model of glioma with an alternative mechanism of telomere maintenance (ALT)
Article Snippet: Briefly, cells were grown as adherent cultures in culture medium supplemented with 10 μM BrdU for one cell cycle. .. Metaphase spreads were stained with Hoechst 33258, exposed to ultraviolet (UV) light, and digested with exonuclease III (Promega). .. Successive hybridizations with a FITC-labeled (TTAGGG)3 PNA probe (Applied Biosystems) and a Cy-3-labeled (CCCTAA)3 PNA probe allowed the detection of the parental telomere C and G strands.

Article Title: Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis
Article Snippet: Slides were stained with Hoechst 33258 (0.50 ng/μL; Sigma-Aldrich) for 15 min and exposed to 365 nm UV light (Stratalinker 2400) for 25 min. .. Following UV exposure, BrdU incorporated strands were digested with Exonuclease III (3 U/μL in provided reaction buffer; Promega) at room temperature for 10 min.

Fluorescence In Situ Hybridization:

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability
Article Snippet: Paragraph title: Cytogenetic analysis and fluorescence in situ hybridization (FISH) staining ... The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

Article Title: Telomeric RNAs are essential to maintain telomeres
Article Snippet: Paragraph title: Chromosome orientation FISH (CO-FISH) ... The slides were treated with 0.5 mg ml−1 RNase A for 10 min at 37 °C, stained with 0.5 μg ml−1 Hoechst 33258 (Sigma) in 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate) for 15 min at room temperature and then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymatic digestion of the BrdU/BrdC-substituted DNA strands with 3 U μl−1 of Exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM dithiothreitol, pH 8) was allowed to proceed for 10 min at room temperature.

Article Title: Human RECQL1 participates in telomere maintenance
Article Snippet: Paragraph title: Chromosome Orientation FISH (CO-FISH) ... Slides were then rinsed with 2× SSC and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 30 min, and then digested the DNA with 10 U/µl exonuclease III (Promega) for 10 min at RT.

Article Title: Functional interaction between DNA-PKcs and telomerase in telomere length maintenance
Article Snippet: Slides were then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymic digestion of the BrdU-substituted DNA strands with 3 U/µl exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM DTT pH 8.0) was allowed to proceed for 10 min at room temperature. .. Slides were then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymic digestion of the BrdU-substituted DNA strands with 3 U/µl exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM DTT pH 8.0) was allowed to proceed for 10 min at room temperature.

Article Title: Telomere Reprogramming and Maintenance in Porcine iPS Cells
Article Snippet: The 5′-bromo-2′-deoxyuridine-substituted DNA was digested with Exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 and 50 mM Tris-HCl, pH 8.0) for 10 min at RT. .. The 5′-bromo-2′-deoxyuridine-substituted DNA was digested with Exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 and 50 mM Tris-HCl, pH 8.0) for 10 min at RT.

Article Title: MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end-resection
Article Snippet: Telomere sister-chromatid exchanges were detected by chromosome-orientation FISH (CO-FISH), essentially as described with minor modifications. .. Following digestion with Exonuclease III (10 U/μl, Promega, Madison, WI) for 10 min at room temperature, slides were dehydrated through an ethanol series (70%, 95% and 100%) and incubated sequentially with Cy3-TelG 5′-[TTAGGG]3 -3′ and FITC-TelC 5′-[CCCTAA]3 -3′ probes at room temperature.

Article Title: Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes
Article Snippet: Strand-Specific FISH (CO-FISH). .. The nicked BrdUrd/dC-substituted DNA was digested with 3 units/μl exonuclease III (Promega) in 50 mM Tris·HCl (pH 8.0), 5 mM MgCl2 , and 5 mM DTT for 10 min at room temperature.

Article Title: Zscan4 regulates telomere elongation and genomic stability in ES cells
Article Snippet: Paragraph title: Telomere Chromosome Orientation FISH (CO-FISH) ... The BrdU-substituted DNA was digested with 3 units/µl Exonuclease III (Promega) for 10 min at room temperature.

Article Title: A Distinct Class of Genome Rearrangements Driven by Heterologous Recombination
Article Snippet: Paragraph title: Chromosome Orientation FISH ... The BrdU-labeled DNA strands were digested with 100 μl Exonuclease III (Promega) at 10 U/ μl, in the buffer supplied by the manufacturer, for 30 min at room temperature.

Article Title: A preclinical mouse model of glioma with an alternative mechanism of telomere maintenance (ALT)
Article Snippet: Chromosome orientation FISH (CO-FISH) was performed as previously described. .. Metaphase spreads were stained with Hoechst 33258, exposed to ultraviolet (UV) light, and digested with exonuclease III (Promega).

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    Promega exonuclease iii
    Overexpression of PRL-3 promotes telomere dysfunction. ( A ) Validation of PRL-3 stable overexpression. WI38 fibroblasts were infected with control or PRL-3-expressing letivirus. Expression vectors pcDNA3-myc-PRL-3 (for HCT116 cells), pcDNA3.1-myc-PRL-3 (for LoVo cells) and the respective control plasmids were transfected into cells, followed by selection and pooling of stable colonies. Cell lysates were examined by western blot with antibodies to PRL-3, TRF2 and RAP1. ( B ) Effects of PRL-3 stable overexpression on γH2AX, pCHK1 and p53 levels. Indicated cells were treated with ETP (20 μM) or DMSO (1:1000) for 4 h. ( C ) Effects of PRL-3 stable overexpression on TIF formation. WI38 cells were analyzed by IF-FISH staining of pATM (green) and telomere (red). Left, representative staining. Arrows, foci of TIFs. Scale bar, 5 μm. Right, quantification of cells with ≥5 TIFs. Mean ± SD of two independent experiments. n > 60 metaphase per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on dysfunctional telomere repair pathways. Upper, representative CO-FISH staining of WI38 cells. Metaphase cells were stained with probes specific for leading (red) and lagging (green) strands and counterstained with DAPI (blue). Yellow arrow, a typical T-SCE. White arrow, a chromosome–chromosome fusion. Red arrowhead, a MTS. Scale bar, 2.5 μm. Lower, quantification of abnormalities. Mean ± SD of two independent experiments. n > 1300 chromosomes per single experiment. Student's t -test. ( E ) Southern blot analysis of PRL-3 stable overexpression-induced telomere deprotection. Genomic <t>DNA</t> from indicated cells were resolved on agarose gel, transferred to nitrocellulose membrane and probed with biotin-labeled telomere probe. ( F ) qPCR analysis of PRL-3 stable overexpression-induced telomere deprotection. Relative telomere to single copy gene (T/S) ratio of control cells was set as 1. Mean ± SD of <t>three</t> independent experiments. n = 4 replicates per single experiment. Student's t -test.
    Exonuclease Iii, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Overexpression of PRL-3 promotes telomere dysfunction. ( A ) Validation of PRL-3 stable overexpression. WI38 fibroblasts were infected with control or PRL-3-expressing letivirus. Expression vectors pcDNA3-myc-PRL-3 (for HCT116 cells), pcDNA3.1-myc-PRL-3 (for LoVo cells) and the respective control plasmids were transfected into cells, followed by selection and pooling of stable colonies. Cell lysates were examined by western blot with antibodies to PRL-3, TRF2 and RAP1. ( B ) Effects of PRL-3 stable overexpression on γH2AX, pCHK1 and p53 levels. Indicated cells were treated with ETP (20 μM) or DMSO (1:1000) for 4 h. ( C ) Effects of PRL-3 stable overexpression on TIF formation. WI38 cells were analyzed by IF-FISH staining of pATM (green) and telomere (red). Left, representative staining. Arrows, foci of TIFs. Scale bar, 5 μm. Right, quantification of cells with ≥5 TIFs. Mean ± SD of two independent experiments. n > 60 metaphase per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on dysfunctional telomere repair pathways. Upper, representative CO-FISH staining of WI38 cells. Metaphase cells were stained with probes specific for leading (red) and lagging (green) strands and counterstained with DAPI (blue). Yellow arrow, a typical T-SCE. White arrow, a chromosome–chromosome fusion. Red arrowhead, a MTS. Scale bar, 2.5 μm. Lower, quantification of abnormalities. Mean ± SD of two independent experiments. n > 1300 chromosomes per single experiment. Student's t -test. ( E ) Southern blot analysis of PRL-3 stable overexpression-induced telomere deprotection. Genomic DNA from indicated cells were resolved on agarose gel, transferred to nitrocellulose membrane and probed with biotin-labeled telomere probe. ( F ) qPCR analysis of PRL-3 stable overexpression-induced telomere deprotection. Relative telomere to single copy gene (T/S) ratio of control cells was set as 1. Mean ± SD of three independent experiments. n = 4 replicates per single experiment. Student's t -test.

    Journal: Nucleic Acids Research

    Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

    doi: 10.1093/nar/gkx392

    Figure Lengend Snippet: Overexpression of PRL-3 promotes telomere dysfunction. ( A ) Validation of PRL-3 stable overexpression. WI38 fibroblasts were infected with control or PRL-3-expressing letivirus. Expression vectors pcDNA3-myc-PRL-3 (for HCT116 cells), pcDNA3.1-myc-PRL-3 (for LoVo cells) and the respective control plasmids were transfected into cells, followed by selection and pooling of stable colonies. Cell lysates were examined by western blot with antibodies to PRL-3, TRF2 and RAP1. ( B ) Effects of PRL-3 stable overexpression on γH2AX, pCHK1 and p53 levels. Indicated cells were treated with ETP (20 μM) or DMSO (1:1000) for 4 h. ( C ) Effects of PRL-3 stable overexpression on TIF formation. WI38 cells were analyzed by IF-FISH staining of pATM (green) and telomere (red). Left, representative staining. Arrows, foci of TIFs. Scale bar, 5 μm. Right, quantification of cells with ≥5 TIFs. Mean ± SD of two independent experiments. n > 60 metaphase per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on dysfunctional telomere repair pathways. Upper, representative CO-FISH staining of WI38 cells. Metaphase cells were stained with probes specific for leading (red) and lagging (green) strands and counterstained with DAPI (blue). Yellow arrow, a typical T-SCE. White arrow, a chromosome–chromosome fusion. Red arrowhead, a MTS. Scale bar, 2.5 μm. Lower, quantification of abnormalities. Mean ± SD of two independent experiments. n > 1300 chromosomes per single experiment. Student's t -test. ( E ) Southern blot analysis of PRL-3 stable overexpression-induced telomere deprotection. Genomic DNA from indicated cells were resolved on agarose gel, transferred to nitrocellulose membrane and probed with biotin-labeled telomere probe. ( F ) qPCR analysis of PRL-3 stable overexpression-induced telomere deprotection. Relative telomere to single copy gene (T/S) ratio of control cells was set as 1. Mean ± SD of three independent experiments. n = 4 replicates per single experiment. Student's t -test.

    Article Snippet: The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

    Techniques: Over Expression, Infection, Expressing, Transfection, Selection, Western Blot, Fluorescence In Situ Hybridization, Staining, Southern Blot, Agarose Gel Electrophoresis, Labeling, Real-time Polymerase Chain Reaction

    Overexpression of PRL-3 promotes chromosomal instability and senescence. ( A ) Effects of PRL-3 stable overexpression on APB and MN formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. Student's t -test. n > 1500 cells scored per sample for MN or n > 60 anaphase cells scored per sample for APB. ( B ) Effects of PRL-3 stable overexpression on BrdU incorporation. Indicated cells were treated with double-thymidine block, released into fresh medium containing 10 μM BrdU and incubated for 45 min. Cells were fixed, immunostained with anti-BrdU (green), and counterstained with DAPI (blue). Left, representative staining of BrdU. Scale bar, 15 μm. Right, quantification of BrdU-positive cells. Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on senescence. Indicated cells were treated with DMSO (1:1000) or Ku55933 (5 μM) for 24 h, followed by β-galactosidase staining. Left, representative staining. Right, quantification of β-galactosidase positive cells. Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on H3K9me3 levels. Indicated cells were fixed, immunostained with anti-H3K9me3 (red), and counterstained with DAPI (blue). ( E ) Effects of reconstituted PRL-3 on telomere length, DNA damage and senescence in PRL-3 stable knockdown cells. HCT116 control and PRL-3 stable knockdown cells were co-transfected with indicated amount of pcDNA3 and pcDNA3-PRL-3 plasmids. The total amount of plasmids for each sample was adjusted to 4 μg. After 72 h, protein lysates were subjected to western blot of PRL-3, γH2AX, H3K9me3 (lower). Genomic DNA was used for qPCR analysis of telomere length (upper). Protein bands were scanned and relative OD was calculated by normalizing to GAPDH. T/S ratio of HCT116 control cells transfected with pcDNA3 was set as 1. Pearson χ2 test.

    Journal: Nucleic Acids Research

    Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

    doi: 10.1093/nar/gkx392

    Figure Lengend Snippet: Overexpression of PRL-3 promotes chromosomal instability and senescence. ( A ) Effects of PRL-3 stable overexpression on APB and MN formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. Student's t -test. n > 1500 cells scored per sample for MN or n > 60 anaphase cells scored per sample for APB. ( B ) Effects of PRL-3 stable overexpression on BrdU incorporation. Indicated cells were treated with double-thymidine block, released into fresh medium containing 10 μM BrdU and incubated for 45 min. Cells were fixed, immunostained with anti-BrdU (green), and counterstained with DAPI (blue). Left, representative staining of BrdU. Scale bar, 15 μm. Right, quantification of BrdU-positive cells. Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on senescence. Indicated cells were treated with DMSO (1:1000) or Ku55933 (5 μM) for 24 h, followed by β-galactosidase staining. Left, representative staining. Right, quantification of β-galactosidase positive cells. Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on H3K9me3 levels. Indicated cells were fixed, immunostained with anti-H3K9me3 (red), and counterstained with DAPI (blue). ( E ) Effects of reconstituted PRL-3 on telomere length, DNA damage and senescence in PRL-3 stable knockdown cells. HCT116 control and PRL-3 stable knockdown cells were co-transfected with indicated amount of pcDNA3 and pcDNA3-PRL-3 plasmids. The total amount of plasmids for each sample was adjusted to 4 μg. After 72 h, protein lysates were subjected to western blot of PRL-3, γH2AX, H3K9me3 (lower). Genomic DNA was used for qPCR analysis of telomere length (upper). Protein bands were scanned and relative OD was calculated by normalizing to GAPDH. T/S ratio of HCT116 control cells transfected with pcDNA3 was set as 1. Pearson χ2 test.

    Article Snippet: The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

    Techniques: Over Expression, Staining, BrdU Incorporation Assay, Blocking Assay, Incubation, Transfection, Western Blot, Real-time Polymerase Chain Reaction

    Silencing of PRL-3 promotes DDR and senescence. ( A ) Efficiencies of PRL-3 silencing in HCT116 (knockdown by two shRNAs using lentivirus system, left) and SW480 (knockout by CRISPR/Cas9 system, right) cells and its effects on indicated protein levels. WT, wild-type. KO, knockout. ( B ) Effects of PRL-3 silencing on phosphorylations of H2AX and CHK1. Samples treated with 20 μM etoposide (ETP) for 4 h were used as positive controls. ( C ) Effects of PRL-3 silencing on TIF formation. Indicated HCT116 cells were subjected to IF-FISH staining. Upper, representative staining. Arrows, colocalizations between γH2AX and telomere (TIFs). Scale bar, 5 μm. Lower, quantification of cells with ≥5 TIF. Mean ± SD of two independent experiments. n > 200 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 silencing on anaphase bridges (APB) and micronuclei (MN) formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. n > 1000 cells scored per sample for MN and n > 50 anaphase cells scored per sample for APB. Student's t -test. Representative images of APB (red arrow) and MN (white arrow) of HCT116 cells stained with DAPI were shown. ( E ) ChIP analysis of RAP1 and TRF2's binding to telomeric or Alu DNA in HCT116 and S480 cells silenced for PRL-3. Upper, representative blots after ChIP with indicated antibodies or IgG. Input, 2% DNA. Lower, quantification of relative OD. Relative OD was calculated by normalizing to that of input and relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( F ) PRL-3 silencing induced ROS-dependent cellular senescence and DNA damage response. Indica ted HCT116 cells were treated with NAC (10 mM), GSH (10 mM) or DMSO (1:1000) for 24 h. Part of cells were fixed and processed for β-galactosidase staining, others were analyzed by western blot. Upper, representative β-galactosidase staining of cells treated with DMSO. Middle, quantification of β-galactosidase positive cells. Mean ± SD of two independent experiments. n > 400 cells per single experiment. Student's t -test. Lower, western blot of γH2AX.

    Journal: Nucleic Acids Research

    Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

    doi: 10.1093/nar/gkx392

    Figure Lengend Snippet: Silencing of PRL-3 promotes DDR and senescence. ( A ) Efficiencies of PRL-3 silencing in HCT116 (knockdown by two shRNAs using lentivirus system, left) and SW480 (knockout by CRISPR/Cas9 system, right) cells and its effects on indicated protein levels. WT, wild-type. KO, knockout. ( B ) Effects of PRL-3 silencing on phosphorylations of H2AX and CHK1. Samples treated with 20 μM etoposide (ETP) for 4 h were used as positive controls. ( C ) Effects of PRL-3 silencing on TIF formation. Indicated HCT116 cells were subjected to IF-FISH staining. Upper, representative staining. Arrows, colocalizations between γH2AX and telomere (TIFs). Scale bar, 5 μm. Lower, quantification of cells with ≥5 TIF. Mean ± SD of two independent experiments. n > 200 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 silencing on anaphase bridges (APB) and micronuclei (MN) formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. n > 1000 cells scored per sample for MN and n > 50 anaphase cells scored per sample for APB. Student's t -test. Representative images of APB (red arrow) and MN (white arrow) of HCT116 cells stained with DAPI were shown. ( E ) ChIP analysis of RAP1 and TRF2's binding to telomeric or Alu DNA in HCT116 and S480 cells silenced for PRL-3. Upper, representative blots after ChIP with indicated antibodies or IgG. Input, 2% DNA. Lower, quantification of relative OD. Relative OD was calculated by normalizing to that of input and relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( F ) PRL-3 silencing induced ROS-dependent cellular senescence and DNA damage response. Indica ted HCT116 cells were treated with NAC (10 mM), GSH (10 mM) or DMSO (1:1000) for 24 h. Part of cells were fixed and processed for β-galactosidase staining, others were analyzed by western blot. Upper, representative β-galactosidase staining of cells treated with DMSO. Middle, quantification of β-galactosidase positive cells. Mean ± SD of two independent experiments. n > 400 cells per single experiment. Student's t -test. Lower, western blot of γH2AX.

    Article Snippet: The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

    Techniques: Knock-Out, CRISPR, Gene Knockout, Fluorescence In Situ Hybridization, Staining, Chromatin Immunoprecipitation, Binding Assay, Western Blot

    RAP1 and TRF2-dependent recruitment of PRL-3 to telomere. ( A ) In situ PLA analysis of PRL-3's associations with RAP1 and TRF2. HCT116 cells were pre-extracted, fixed, inmunostained with indicated pairs of antibodies and probed with Duolink in situ PLA reagent. Binding foci were in red and dashed lines indicated outline of nucleus (determined by DAPI counter staining). Scale bar, 10 μm. ( B ) TRF2- and RAP1-dependent recruitment of PRL-3 to telomeric DNA in vitro . Purified myc-TRF2 (150 ng), His-RAP1 (120 ng), and His-PRL-3 (30 ng) were co-incubated with 1 μg biotin-labeled telomere (lanes 1–4) or Alu (lanes 5–8) probe as indicated and subjected to pull-down analysis with Streptavidin agarose. Precipitates were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. ( C and D ) TRF2 and RAP1-dependent recruitment of PRL-3 to telomere in cells. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h, pre-extracted, fixed and subjected to IF-FISH staining. (C) Representative PRL-3 association with telomere. Scale bar, 10 μm. Areas in white squares were enlarged. (D) Quantification of cells with ≥5 associations between PRL-3 foci and telomere. Mean ± SD of three independent experiments. n > 100 cells per single experiment. Student's t -test. ( E ) Knockdown efficiencies of RAP1 and TRF2. HCT116 cells were transfected with 50 nM siRNAs against RAP1 or TRF2 for 48 h. Lysates were analyzed by western blot with indicated antibodies. ( F ) ChIP analysis of PRL-3 binding to telomeric and Alu DNA. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h and processed for ChIP using anti-PRL-3 or pre-immune IgG. Upper, representative blots of hybridization with probe to telomere or Alu. Input, 2% DNA. Lower, quantification of relative optical densities (OD). Relative OD was calculated by normalizing to OD of Input and relative OD of control siRNA-transfected sample was set as 100%. Mean ± SD of three independent experiments. Student's t -test.

    Journal: Nucleic Acids Research

    Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

    doi: 10.1093/nar/gkx392

    Figure Lengend Snippet: RAP1 and TRF2-dependent recruitment of PRL-3 to telomere. ( A ) In situ PLA analysis of PRL-3's associations with RAP1 and TRF2. HCT116 cells were pre-extracted, fixed, inmunostained with indicated pairs of antibodies and probed with Duolink in situ PLA reagent. Binding foci were in red and dashed lines indicated outline of nucleus (determined by DAPI counter staining). Scale bar, 10 μm. ( B ) TRF2- and RAP1-dependent recruitment of PRL-3 to telomeric DNA in vitro . Purified myc-TRF2 (150 ng), His-RAP1 (120 ng), and His-PRL-3 (30 ng) were co-incubated with 1 μg biotin-labeled telomere (lanes 1–4) or Alu (lanes 5–8) probe as indicated and subjected to pull-down analysis with Streptavidin agarose. Precipitates were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. ( C and D ) TRF2 and RAP1-dependent recruitment of PRL-3 to telomere in cells. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h, pre-extracted, fixed and subjected to IF-FISH staining. (C) Representative PRL-3 association with telomere. Scale bar, 10 μm. Areas in white squares were enlarged. (D) Quantification of cells with ≥5 associations between PRL-3 foci and telomere. Mean ± SD of three independent experiments. n > 100 cells per single experiment. Student's t -test. ( E ) Knockdown efficiencies of RAP1 and TRF2. HCT116 cells were transfected with 50 nM siRNAs against RAP1 or TRF2 for 48 h. Lysates were analyzed by western blot with indicated antibodies. ( F ) ChIP analysis of PRL-3 binding to telomeric and Alu DNA. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h and processed for ChIP using anti-PRL-3 or pre-immune IgG. Upper, representative blots of hybridization with probe to telomere or Alu. Input, 2% DNA. Lower, quantification of relative optical densities (OD). Relative OD was calculated by normalizing to OD of Input and relative OD of control siRNA-transfected sample was set as 100%. Mean ± SD of three independent experiments. Student's t -test.

    Article Snippet: The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

    Techniques: In Situ, Proximity Ligation Assay, Binding Assay, Staining, In Vitro, Purification, Incubation, Labeling, Western Blot, Transfection, Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Hybridization

    PRL-3 relocates RAP1 and TRF2 from telomeric DNA. ( A ) Effects of PRL-3 stable overexpression on the chromatin abundance of RAP1, TRF2 and TRF1. Nuclei from HCT116 cells were homogenized in buffer containing indicated concentrations of NaCl. Chromatin-enriched fractions were analyzed by western blot. Left, representative blots. Right, relative levels of TRF2, RAP1 and TRF1. Protein band were scanned and relative OD was calculated by normalizing to OD of H2B. The relative OD of sample prepared with 150 mM NaCl was set as 100%. Mean ± SD of three independent experiments. ANOVA. ( B ) Effects of PRL-3 stable overexpression on bindings of RAP1 and TRF2 to telomeric and Alu DNA. Indicated cells were crosslinked, immunoprecipitated with antibodies to RAP1, TRF2 or pre-immune IgG, and precipitated DNA was analyzed by ChIP. Upper, representative blots. Lower, quantification of relative OD, which was calculated by normalizing to that of Input. Relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on telomere associations of RAP1 and TRF2 in WI38 cells. Left, representative IF-FISH staining of telomere (red) and RAP1 or TRF2 (green). Arrows, foci of co-localization. Scale bar, 10 μm. Right, quantification of cells with ≥5 associations between RAP1 or TRF2 foci and telomere. Mean ± SD of two independent experiments. n > 80 cells per single experiment. Student's t -test. ( D ) EMSA analysis of PRL-3, RAP1 and TRF2's associations with telomeric DNA. Indicated concentrations of purified FLAG-TRF2, His-RAP1, myc-PRL-3 were co-incubated with Biotin-labeled telomere probe (20 nM). To induce super-shift, 0.1 μg anti-PRL-3 (lane 5), anti-TRF2 (lanes 6 and 18) and IgG (lane 7) were used. Note that anti-PRL-3 and anti-TRF2-induced super-shifts of Complex II partially co-migrated with Complex I (lanes 5 and 6).

    Journal: Nucleic Acids Research

    Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

    doi: 10.1093/nar/gkx392

    Figure Lengend Snippet: PRL-3 relocates RAP1 and TRF2 from telomeric DNA. ( A ) Effects of PRL-3 stable overexpression on the chromatin abundance of RAP1, TRF2 and TRF1. Nuclei from HCT116 cells were homogenized in buffer containing indicated concentrations of NaCl. Chromatin-enriched fractions were analyzed by western blot. Left, representative blots. Right, relative levels of TRF2, RAP1 and TRF1. Protein band were scanned and relative OD was calculated by normalizing to OD of H2B. The relative OD of sample prepared with 150 mM NaCl was set as 100%. Mean ± SD of three independent experiments. ANOVA. ( B ) Effects of PRL-3 stable overexpression on bindings of RAP1 and TRF2 to telomeric and Alu DNA. Indicated cells were crosslinked, immunoprecipitated with antibodies to RAP1, TRF2 or pre-immune IgG, and precipitated DNA was analyzed by ChIP. Upper, representative blots. Lower, quantification of relative OD, which was calculated by normalizing to that of Input. Relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on telomere associations of RAP1 and TRF2 in WI38 cells. Left, representative IF-FISH staining of telomere (red) and RAP1 or TRF2 (green). Arrows, foci of co-localization. Scale bar, 10 μm. Right, quantification of cells with ≥5 associations between RAP1 or TRF2 foci and telomere. Mean ± SD of two independent experiments. n > 80 cells per single experiment. Student's t -test. ( D ) EMSA analysis of PRL-3, RAP1 and TRF2's associations with telomeric DNA. Indicated concentrations of purified FLAG-TRF2, His-RAP1, myc-PRL-3 were co-incubated with Biotin-labeled telomere probe (20 nM). To induce super-shift, 0.1 μg anti-PRL-3 (lane 5), anti-TRF2 (lanes 6 and 18) and IgG (lane 7) were used. Note that anti-PRL-3 and anti-TRF2-induced super-shifts of Complex II partially co-migrated with Complex I (lanes 5 and 6).

    Article Snippet: The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

    Techniques: Over Expression, Western Blot, Immunoprecipitation, Chromatin Immunoprecipitation, Fluorescence In Situ Hybridization, Staining, Purification, Incubation, Labeling

    Disrupting PRL-3-RAP1 complex or expressing ectopic TRF2 attenuates PRL-3 overexpression-promoted telomere deprotection, DNA damage, chromosomal instability and senescence. ( A ) HCT116 control and PRL-3 overexpressing cells were transfected with 0.5 μg of pEGFP-N1-Myb or pEGFP-N1 plasmid for 72 h, and indicated proteins were analyzed by western blot. ( B ) qPCR analysis of telomere length of cells in (A). T/S ratio of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test. ( C ) Quantification of micronuclei of cells in (A). Mean ± SD of two independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Quantification of β-galactosidase-positive cells in (A). Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( E ) Relative migration of cells in (A). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of two independent experiments. Three replicates per single experiment. Student's t -test. ( F ) HCT116 control and PRL-3 overexpressing cells were infected with control (Lv-con) or TRF2-expressing lentivirus (Lv-TRF2) for 120 h, and lysates were subjected to western blot. ( G ) qPCR analysis of telomere length of cells in (F). T/S ratio of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. 3 replicates per single experiment. Student's t -test. ( H ) Quantification of micronuclei of cells in (F). Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( I ) Quantification of β-galactosidase-positive cells in (F). Mean ± SD of three independent experiments. n > 300 cells per single experiment. Student's t -test. ( J ) Relative migration of cells of (F). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test.

    Journal: Nucleic Acids Research

    Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

    doi: 10.1093/nar/gkx392

    Figure Lengend Snippet: Disrupting PRL-3-RAP1 complex or expressing ectopic TRF2 attenuates PRL-3 overexpression-promoted telomere deprotection, DNA damage, chromosomal instability and senescence. ( A ) HCT116 control and PRL-3 overexpressing cells were transfected with 0.5 μg of pEGFP-N1-Myb or pEGFP-N1 plasmid for 72 h, and indicated proteins were analyzed by western blot. ( B ) qPCR analysis of telomere length of cells in (A). T/S ratio of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test. ( C ) Quantification of micronuclei of cells in (A). Mean ± SD of two independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Quantification of β-galactosidase-positive cells in (A). Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( E ) Relative migration of cells in (A). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of two independent experiments. Three replicates per single experiment. Student's t -test. ( F ) HCT116 control and PRL-3 overexpressing cells were infected with control (Lv-con) or TRF2-expressing lentivirus (Lv-TRF2) for 120 h, and lysates were subjected to western blot. ( G ) qPCR analysis of telomere length of cells in (F). T/S ratio of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. 3 replicates per single experiment. Student's t -test. ( H ) Quantification of micronuclei of cells in (F). Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( I ) Quantification of β-galactosidase-positive cells in (F). Mean ± SD of three independent experiments. n > 300 cells per single experiment. Student's t -test. ( J ) Relative migration of cells of (F). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test.

    Article Snippet: The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

    Techniques: Expressing, Over Expression, Transfection, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Migration, Infection

    Conservative DNA replication at telomeres of human ALT cells Examples of chromosome arms exhibiting features of telomeric semiconservative (Semi) replication, conservative (Consrv) replication of the entire telomere (E) and conservative replication of part of the telomere (P). Idealized chromosome diagrams (left) and actual microscopy images (right) are shown. D‐FISH, denaturing FISH; ND‐CO‐FISH, non‐denaturing CO‐FISH. Percentages of chromosome arms exhibiting conservative (Consrv) replication of the entire telomere (E), conservative replication of part of the telomere (P), and conservative replication of either the entire telomere of part of it (E+P). NE, U2OS cells expressing normal levels of cyclin E; OE, U2OS cells overexpressing cyclin E for 4 days. Bars represent means and standard errors of the mean from three independent experiments. Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres: P

    Journal: EMBO Reports

    Article Title: Alternative lengthening of human telomeres is a conservative DNA replication process with features of break‐induced replication

    doi: 10.15252/embr.201643169

    Figure Lengend Snippet: Conservative DNA replication at telomeres of human ALT cells Examples of chromosome arms exhibiting features of telomeric semiconservative (Semi) replication, conservative (Consrv) replication of the entire telomere (E) and conservative replication of part of the telomere (P). Idealized chromosome diagrams (left) and actual microscopy images (right) are shown. D‐FISH, denaturing FISH; ND‐CO‐FISH, non‐denaturing CO‐FISH. Percentages of chromosome arms exhibiting conservative (Consrv) replication of the entire telomere (E), conservative replication of part of the telomere (P), and conservative replication of either the entire telomere of part of it (E+P). NE, U2OS cells expressing normal levels of cyclin E; OE, U2OS cells overexpressing cyclin E for 4 days. Bars represent means and standard errors of the mean from three independent experiments. Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres: P

    Article Snippet: The 5′‐bromo‐2′‐deoxyuridine‐ and 5′‐bromo‐2′‐deoxycytosine‐substituted DNA was digested with exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 , and 50 mM Tris–HCl, pH 8.0) for 15 min at 37°C.

    Techniques: Microscopy, Fluorescence In Situ Hybridization, Expressing, Over Expression

    Triple‐FISH protocol to distinguish between telomeric semiconservative and conservative DNA replication Semiconservative replication. Diagram of a chromosome with the telomeric C‐rich and G‐rich strands colored red and green, respectively. Newly synthesized strands are indicated by dotted lines. The three steps of the protocol were strand‐specific: dual color, denaturing FISH (1), non‐denaturing chromosome orientation (CO)‐FISH (2), and denaturing FISH (3). In the second step, the nascent strands have been digested. In all steps, two sets of PNA primers specific for the G‐strand and C‐strand, respectively, were used to monitor the presence of both strands. The arrows indicate the color of the emitted light and its intensity (idealized). Conservative replication. Diagram showing that telomeric conservative replication (shown here to involve the entire length of the telomeres at the p arms) leads to distinct staining patterns, not observed with semiconservative replication. BIR, break‐induced replication. Partially conservative and partially semiconservative telomeric replication. Diagram showing the staining patterns predicted for telomeres that are partially conservatively replicated (distal half) and partially semiconservatively replicated (proximal half), as might occur following fork collapse within a telomere (shown only for the telomeres at the p arms).

    Journal: EMBO Reports

    Article Title: Alternative lengthening of human telomeres is a conservative DNA replication process with features of break‐induced replication

    doi: 10.15252/embr.201643169

    Figure Lengend Snippet: Triple‐FISH protocol to distinguish between telomeric semiconservative and conservative DNA replication Semiconservative replication. Diagram of a chromosome with the telomeric C‐rich and G‐rich strands colored red and green, respectively. Newly synthesized strands are indicated by dotted lines. The three steps of the protocol were strand‐specific: dual color, denaturing FISH (1), non‐denaturing chromosome orientation (CO)‐FISH (2), and denaturing FISH (3). In the second step, the nascent strands have been digested. In all steps, two sets of PNA primers specific for the G‐strand and C‐strand, respectively, were used to monitor the presence of both strands. The arrows indicate the color of the emitted light and its intensity (idealized). Conservative replication. Diagram showing that telomeric conservative replication (shown here to involve the entire length of the telomeres at the p arms) leads to distinct staining patterns, not observed with semiconservative replication. BIR, break‐induced replication. Partially conservative and partially semiconservative telomeric replication. Diagram showing the staining patterns predicted for telomeres that are partially conservatively replicated (distal half) and partially semiconservatively replicated (proximal half), as might occur following fork collapse within a telomere (shown only for the telomeres at the p arms).

    Article Snippet: The 5′‐bromo‐2′‐deoxyuridine‐ and 5′‐bromo‐2′‐deoxycytosine‐substituted DNA was digested with exonuclease III (Promega) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2 , and 50 mM Tris–HCl, pH 8.0) for 15 min at 37°C.

    Techniques: Fluorescence In Situ Hybridization, Synthesized, Staining

    Deletion of the 20q-TERRA locus decreases telomere length and protection in U2OS cells. ( a ) Q-FISH images obtained from metaphases spreads from U2OS cells WT and KO for the Chr20q-TERRA locus (clones A4, B4 and C4). (Left graphs) Frequency graphs of telomere length (a.u.) distribution measured in WT and in the 20q-KO cells (clones A4, B4 and C4) from three independent experiments. The mean telomere length and the number of telomeres and metaphases analyzed is shown. The red lines are arbitrary lines placed in the exact same position in each frequency graph to visualize differences between the 20q-KO clones and the WT controls (right graphs) The mean telomere length, the percentage of short telomeres and the quantification of signal-free ends per metaphase are also represented. Short telomeres are considered those in the 10% percentile of the total telomere length distribution. Total number of metaphases used for the statistical analysis is indicated. Scale bar, 10 μm and (zoom) 1 μm. ( b ) WT and 20q-KO cells were analyzed for T-SCE events with G-rich (green) and C-rich (red) PNA probes. The fraction of chromosome ends with T-SCE obtained from three different experiments was quantified and graphed as the mean values±s.e.m., n =30 metaphases. The number of metaphases analyzed is shown. Only events in which interchange of both colours were quantified (see examples of no-T-SCE and T-SCE). The quantification was carried out by counting the number of events in the same chromosome or in different chromosomes and then normalizing it by the total number of chromosomes observed in each metaphase. Scale bar, 1 μm. ( c ) Quantification of DNA-containing double minute chromosomes (TDMs) in WT and 20q-KO cells from three different experiments (mean values±s.e.m., n =30 metaphases). An example of TDMs is shown. One-way Anova with Dunnett's post test was used for all statistical analysis (* P

    Journal: Nature Communications

    Article Title: Telomeric RNAs are essential to maintain telomeres

    doi: 10.1038/ncomms12534

    Figure Lengend Snippet: Deletion of the 20q-TERRA locus decreases telomere length and protection in U2OS cells. ( a ) Q-FISH images obtained from metaphases spreads from U2OS cells WT and KO for the Chr20q-TERRA locus (clones A4, B4 and C4). (Left graphs) Frequency graphs of telomere length (a.u.) distribution measured in WT and in the 20q-KO cells (clones A4, B4 and C4) from three independent experiments. The mean telomere length and the number of telomeres and metaphases analyzed is shown. The red lines are arbitrary lines placed in the exact same position in each frequency graph to visualize differences between the 20q-KO clones and the WT controls (right graphs) The mean telomere length, the percentage of short telomeres and the quantification of signal-free ends per metaphase are also represented. Short telomeres are considered those in the 10% percentile of the total telomere length distribution. Total number of metaphases used for the statistical analysis is indicated. Scale bar, 10 μm and (zoom) 1 μm. ( b ) WT and 20q-KO cells were analyzed for T-SCE events with G-rich (green) and C-rich (red) PNA probes. The fraction of chromosome ends with T-SCE obtained from three different experiments was quantified and graphed as the mean values±s.e.m., n =30 metaphases. The number of metaphases analyzed is shown. Only events in which interchange of both colours were quantified (see examples of no-T-SCE and T-SCE). The quantification was carried out by counting the number of events in the same chromosome or in different chromosomes and then normalizing it by the total number of chromosomes observed in each metaphase. Scale bar, 1 μm. ( c ) Quantification of DNA-containing double minute chromosomes (TDMs) in WT and 20q-KO cells from three different experiments (mean values±s.e.m., n =30 metaphases). An example of TDMs is shown. One-way Anova with Dunnett's post test was used for all statistical analysis (* P

    Article Snippet: The slides were treated with 0.5 mg ml−1 RNase A for 10 min at 37 °C, stained with 0.5 μg ml−1 Hoechst 33258 (Sigma) in 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate) for 15 min at room temperature and then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymatic digestion of the BrdU/BrdC-substituted DNA strands with 3 U μl−1 of Exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM dithiothreitol, pH 8) was allowed to proceed for 10 min at room temperature.

    Techniques: Fluorescence In Situ Hybridization, Gene Knockout, Clone Assay

    Deletion of the 20q-TERRA locus decreases telomere protection in U2OS cells. ( a ) Quantification of the total γH2AX signal per nucleus (mean values±s.e.m., n =number of cells) is shown. The total number of cells analyzed is indicated. ( b ) Quantification of the total 53BP1 spot signal per nucleus (mean values±s.e.m., n =number of cells is shown). The total number of cells analyzed is indicated. ( c ) Graphs showing the quantification of the co-localization (TIF) between TRF2 and either γH2AX or 53BP1 in WT cells and in all 20q-KO clones (mean values±s.e.m., n =3 independent experiments for γH2AX and n =number of cells for 53BP1) per cell is shown. The total number of nuclei analyzed is indicated. ( d ) Representative images of the average number of TIFs found on double inmunostain to detect the telomere protein TRF2 (green) and either the DNA damage markers phospho-Histone γH2AX or 53BP1 (red) in the U2OS cells WT or deleted for the 20q locus. Arrowheads indicate co-localization events. Scale bar, 10 μm. ( e ) Quantification of chromosomal end-to-end fusions in WT and in the 20q-KO cells from three independent experiments (mean values±s.e.m., n =metaphases). Examples of end-to-end fusions are shown as well. Scale bar, 1 μm. ( f ) Array-CGH analysis was performed on hybridization on the same membrane of DNA differentially labelled from WT and 20q-KO cells. The chromosomal gains and losses in 20q-KO cells normalized by WT cells are represented. The chromosomal gains are shown in green and in red the chromosomal losses. One-way Anova with Dunnett's post test was used for all statistical analysis except for the quantification of chromosomal fusions in which the Student's t -test was used (* P

    Journal: Nature Communications

    Article Title: Telomeric RNAs are essential to maintain telomeres

    doi: 10.1038/ncomms12534

    Figure Lengend Snippet: Deletion of the 20q-TERRA locus decreases telomere protection in U2OS cells. ( a ) Quantification of the total γH2AX signal per nucleus (mean values±s.e.m., n =number of cells) is shown. The total number of cells analyzed is indicated. ( b ) Quantification of the total 53BP1 spot signal per nucleus (mean values±s.e.m., n =number of cells is shown). The total number of cells analyzed is indicated. ( c ) Graphs showing the quantification of the co-localization (TIF) between TRF2 and either γH2AX or 53BP1 in WT cells and in all 20q-KO clones (mean values±s.e.m., n =3 independent experiments for γH2AX and n =number of cells for 53BP1) per cell is shown. The total number of nuclei analyzed is indicated. ( d ) Representative images of the average number of TIFs found on double inmunostain to detect the telomere protein TRF2 (green) and either the DNA damage markers phospho-Histone γH2AX or 53BP1 (red) in the U2OS cells WT or deleted for the 20q locus. Arrowheads indicate co-localization events. Scale bar, 10 μm. ( e ) Quantification of chromosomal end-to-end fusions in WT and in the 20q-KO cells from three independent experiments (mean values±s.e.m., n =metaphases). Examples of end-to-end fusions are shown as well. Scale bar, 1 μm. ( f ) Array-CGH analysis was performed on hybridization on the same membrane of DNA differentially labelled from WT and 20q-KO cells. The chromosomal gains and losses in 20q-KO cells normalized by WT cells are represented. The chromosomal gains are shown in green and in red the chromosomal losses. One-way Anova with Dunnett's post test was used for all statistical analysis except for the quantification of chromosomal fusions in which the Student's t -test was used (* P

    Article Snippet: The slides were treated with 0.5 mg ml−1 RNase A for 10 min at 37 °C, stained with 0.5 μg ml−1 Hoechst 33258 (Sigma) in 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate) for 15 min at room temperature and then exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 25–30 min. Enzymatic digestion of the BrdU/BrdC-substituted DNA strands with 3 U μl−1 of Exonuclease III (Promega) in buffer supplied by the manufacturer (50 mM Tris–HCl, 5 mM MgCl2 and 5 mM dithiothreitol, pH 8) was allowed to proceed for 10 min at room temperature.

    Techniques: Gene Knockout, Clone Assay, Hybridization

    RECQL1 associates to telomeres in ALT cells. ( A ) Telomeric ChIP was performed in U2OS cells using anti-RECQL1 antibody in the presence and absence of HU (treated with 5 mM for 18 h). The precipitated DNA was hybridized with the sty-11 telomeric probe (top) or an Alu probe (bottom). Dot blots were performed using anti-RECQL1 antibody, normal IgG and anti-TRF1 antibody that is used as a positive control. ( B ) Telomeric ChIP samples were confirmed by western blotting for the presence of RECQL1 and TRF1. ( C ) The graph showing the signal/input ratio from the RECQL1 IPs as a function of the quantity of chromatin (input). The results were visualized by Phosphor Imager and quantitated with Image Quant software (Molecular Dynamics) and are expressed in % binding to the telomeres. The error bars represent the mean and standard deviation from three independent ChIP assays.

    Journal: Nucleic Acids Research

    Article Title: Human RECQL1 participates in telomere maintenance

    doi: 10.1093/nar/gku200

    Figure Lengend Snippet: RECQL1 associates to telomeres in ALT cells. ( A ) Telomeric ChIP was performed in U2OS cells using anti-RECQL1 antibody in the presence and absence of HU (treated with 5 mM for 18 h). The precipitated DNA was hybridized with the sty-11 telomeric probe (top) or an Alu probe (bottom). Dot blots were performed using anti-RECQL1 antibody, normal IgG and anti-TRF1 antibody that is used as a positive control. ( B ) Telomeric ChIP samples were confirmed by western blotting for the presence of RECQL1 and TRF1. ( C ) The graph showing the signal/input ratio from the RECQL1 IPs as a function of the quantity of chromatin (input). The results were visualized by Phosphor Imager and quantitated with Image Quant software (Molecular Dynamics) and are expressed in % binding to the telomeres. The error bars represent the mean and standard deviation from three independent ChIP assays.

    Article Snippet: Slides were then rinsed with 2× SSC and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) for 30 min, and then digested the DNA with 10 U/µl exonuclease III (Promega) for 10 min at RT.

    Techniques: Chromatin Immunoprecipitation, Positive Control, Western Blot, Software, Binding Assay, Standard Deviation