Journal: Oncoimmunology

Article Title: Association of TRF2 expression and myeloid-derived suppressor cells infiltration with clinical outcome of patients with cutaneous melanoma

doi: 10.1080/2162402X.2021.1901446

Figure Lengend Snippet: Representative images of immune biomarkers and TRF2 staining, and their cell detection mask overlays used in the digital image analysis. Original magnification, x 200

Article Snippet: Formalin-fixed paraffin-embedded (FFPE) serial 4 μm tissue sections were freshly cut, deparaffinized, pre-treated, and stained with monoclonal antibodies (Abs) directed against CD33 (clone SP266, ready-to-use, Roche, Tucson, AZ, USA), CD14 (clone EP128, dilution 1/200, Epitomics, Burlingame, CA, USA), CD15 (clone MMA, ready-to-use, Roche, Tucson, AZ, USA), and TRF2 (clone 4A794.15, dilution 1/500, OriGene, Rockville, MA, USA) on a BenchMark ULTRA autostainer (Ventana Medical Systems, Tucson, AZ, USA).

Techniques: Staining

Journal: Oncoimmunology

Article Title: Association of TRF2 expression and myeloid-derived suppressor cells infiltration with clinical outcome of patients with cutaneous melanoma

doi: 10.1080/2162402X.2021.1901446

Figure Lengend Snippet: Correlative analysis between the clinical and histomolecular characteristics of the patients and the analyzed biomarkers in the metastatic melanoma cohort. *χ2-test, Student’s t-test or ANOVA test were used to investigate difference between groups.

Article Snippet: Formalin-fixed paraffin-embedded (FFPE) serial 4 μm tissue sections were freshly cut, deparaffinized, pre-treated, and stained with monoclonal antibodies (Abs) directed against CD33 (clone SP266, ready-to-use, Roche, Tucson, AZ, USA), CD14 (clone EP128, dilution 1/200, Epitomics, Burlingame, CA, USA), CD15 (clone MMA, ready-to-use, Roche, Tucson, AZ, USA), and TRF2 (clone 4A794.15, dilution 1/500, OriGene, Rockville, MA, USA) on a BenchMark ULTRA autostainer (Ventana Medical Systems, Tucson, AZ, USA).

Techniques: Biomarker Discovery, Mutagenesis

Journal: Oncoimmunology

Article Title: Association of TRF2 expression and myeloid-derived suppressor cells infiltration with clinical outcome of patients with cutaneous melanoma

doi: 10.1080/2162402X.2021.1901446

Figure Lengend Snippet: Multivariate analysis for overall survival in the cohort population

Article Snippet: Formalin-fixed paraffin-embedded (FFPE) serial 4 μm tissue sections were freshly cut, deparaffinized, pre-treated, and stained with monoclonal antibodies (Abs) directed against CD33 (clone SP266, ready-to-use, Roche, Tucson, AZ, USA), CD14 (clone EP128, dilution 1/200, Epitomics, Burlingame, CA, USA), CD15 (clone MMA, ready-to-use, Roche, Tucson, AZ, USA), and TRF2 (clone 4A794.15, dilution 1/500, OriGene, Rockville, MA, USA) on a BenchMark ULTRA autostainer (Ventana Medical Systems, Tucson, AZ, USA).

Techniques: Biomarker Discovery

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkx392

Figure Lengend Snippet: RAP1 mediates PRL-3–TRF2 interaction. ( A ) Precipitation of endogenous RAP1 and TRF2 by GST-PRL-3. HCT116 cell lysates (500 μg) were co-incubated with 1 μg purified GST (lane 2) or GST-PRL-3 (lane 3), and subjected to GST pull-down with Glutathione-agarose beads. Precipitates and 50 μg HCT116 cell lysates (lane 1, input) were analyzed by western blot with indicated anti-shelterin antibodies. Purities of GST-PRL-3 and GST were verified by Coomassie blue staining (lower panel). ( B ) Endogenous PRL-3 associates with RAP1 and TRF2 in cells in a DNA/RNA-independent manner. HCT116 cell lysates (500 μg) were immunoprecipitated by 1 μg antibody against PRL-3 (upper panel) or RAP1 (lower panel). For control, 1 μg preimmune IgG was used. Parts of lysates were also pre-treated with benzonase (Benz) for 30 min at room temperature before immunoprecipitation. Precipitates and 25 μg HCT116 cell lysates (input) were subjected to western blot. ( C ) Requirement of RAP1 for PRL-3-TRF2 association in vitro . Purified proteins (100 ng each) were mixed as indicated (lanes 4–11) and subjected to GST pull-down assay. Some of mixtures were also pre-treated with benzonase for 30 min at room temperature (lanes 8–11). Precipitates and purified proteins (10 ng each, lanes 1–3, input) were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. ( D ) Enhancement of PRL-3–TRF2 interaction by RAP1 in cells. COS7 cells were co-transfected with indicated amounts of pcDNA3 and pcDNA3-myc-RAP1 plasmids. The total amount of plasmids for each sample was adjusted to 2 μg. After 48 h, cells were harvested and lysates were immunoprecipitated with anti-PRL-3 and analyzed by western blot with antibodies to TRF2, myc-tag, RAP1 and PRL-3. ( E ) Requirement of RAP1 for PRL-3–TRF2 association in cells. HCT116 cells were transfected with 50 nM control or RAP1-specific siRNA for 48 h. Cell lysates were immunoprecipitated with anti-PRL-3. HC, IgG heavy chain. ( F ) Upper, GST pull-down assay to map the domain of RAP1 required for its interaction with PRL-3. A total of 100 ng GST (lane 2) or GST-RAP1s (lanes 3–8) was co-incubated with 100 ng His-PRL-3 (lanes 2–8). After pull-down, precipitates were detected by anti-PRL3 and anti-GST. Input, 10 ng His-PRL-3 (lane 1). Lower, summary of binding. FL, full-length RAP1; ΔB, deletion of BRCT domain; ΔBΔM, deletion of BRCT and Myb domains; ΔCΔRΔN, deletion of coiled-coil, RCT and NLS domains. Red asterisks, position of GST or GST fusion proteins. ( G ) Adaptor function of RAP1 in mediating TRF2 and PRL-3 interaction is dependent on its Myb and RCT domains. Purified FALG-TRF2, GST-RAP1 (FL, ΔBΔM, ΔCΔRΔN) and His-PRL-3 proteins (100 ng each) were mixed as indicated. Five percent of mixtures were kept as input, and the rests were subjected to pull-down with anti-FLAG-agarose bead. Precipitates and input were analyzed by western blot with antibodies to PRL-3, TRF2 and GST-tag. ( H ) Blockade of PRL-3's recruitment to RAP1–TRF2 complex by GFP-Myb. HCT116 cells were transfected with 0.5 μg of pEGFP-N1 or pEGFP-N1-Myb plasmid for 48 h. Lysates (500 μg) were immunoprecipitated with 1 μg anti-RAP1 or pre-immune IgG. Precipitates and 25 μg lysates (input) were analyzed by western blot.

Article Snippet: Recombinant FLAG-TRF2, myc-TRF2 and myc-PRL-3 (all from OriGene) were expressed in human HEK293 cells and purified.

Techniques: Incubation, Purification, Western Blot, Staining, Immunoprecipitation, In Vitro, Pull Down Assay, Transfection, Binding Assay, Plasmid Preparation

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: Recombinant FLAG-TRF2, myc-TRF2 and myc-PRL-3 (all from OriGene) were expressed in human HEK293 cells and purified.

Techniques: In Situ, Binding Assay, Staining, In Vitro, Purification, Incubation, Labeling, Western Blot, Transfection, Hybridization

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. Indicated 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: Recombinant FLAG-TRF2, myc-TRF2 and myc-PRL-3 (all from OriGene) were expressed in human HEK293 cells and purified.

Techniques: Knock-Out, CRISPR, Staining, Binding Assay, Western Blot

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: Recombinant FLAG-TRF2, myc-TRF2 and myc-PRL-3 (all from OriGene) were expressed in human HEK293 cells and purified.

Techniques: Over Expression, Infection, Expressing, Transfection, Selection, Western Blot, Staining, Southern Blot, Agarose Gel Electrophoresis, Labeling

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: Recombinant FLAG-TRF2, myc-TRF2 and myc-PRL-3 (all from OriGene) were expressed in human HEK293 cells and purified.

Techniques: Over Expression, Western Blot, Immunoprecipitation, Staining, Purification, Incubation, Labeling

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: Recombinant FLAG-TRF2, myc-TRF2 and myc-PRL-3 (all from OriGene) were expressed in human HEK293 cells and purified.

Techniques: Expressing, Over Expression, Transfection, Plasmid Preparation, Western Blot, Migration, Infection

Journal: Nature Communications

Article Title: Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions

doi: 10.1038/ncomms10132

Figure Lengend Snippet: ( a ) Elevated levels of TRF2 protein in a number of breast cancer and melanoma cells. Immunoblotting was performed to detect TRF2 in whole-cell extracts of the following human cell lines: Primary fibroblasts: IMR90, BJ and WI38; Breast cancer cells: MDA-MB-231, MDA-MB-453, MDA-MB-468, ZR-75-1, MCF-7 and SK-BR-3; Melanoma cells: Lox, CaCL 73-36, WM115, WM278, WM983A, WM983B and WM1158. Fibrosarcoma cell: HT1080. Tubulin was used as a loading control. ( b ) Assessing TRF2 overexpression levels. Parallel cultures of HT1080 clone A6 (a subclone of HT1080 cells that maintain stable telomere length) cells infected with lentiviruses expressing GFP or TRF2 were examined by immunoblotting (top panel) or immunostaining (bottom panel). Fold of TRF2 expression was quantified by the ImageJ software and normalized to tubulin levels. ( c ) Terminal Restriction Fragment analysis of HT1080 A6 cells infected with lentiviruses expressing GFP or TRF2. Cells were continuously passaged and collected at the indicated population doublings (PD). ( d ) Schematic diagram of STELA analysis. ( e ) Individual telomere lengths measured by STELA analysis in HT1080 A6 cells overexpressing GFP or TRF2 at PD6. Each lane represents a single PCR reaction performed with 100 pg of genomic DNA, followed by Southern blotting detection of XpYp telomeres using an XpYp subtelomeric probe.

Article Snippet: Immunoblots were incubated with a mouse monoclonal anti-TRF2 (BD Transduction Laboratories, 1:500), followed by a horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunResearch).

Techniques: Western Blot, Over Expression, Infection, Expressing, Immunostaining, Software, Southern Blot

Journal: Nature Communications

Article Title: Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions

doi: 10.1038/ncomms10132

Figure Lengend Snippet: ( a ) Representative metaphase spread image of HeLa1.2.11 cells infected with lentivirus expressing GFP or TRF2. Infected cells were passaged and collected at PD7 for metaphase spread followed by FISH analysis. Chromosomes (blue) were hybridized with PNA probes for telomeric sequences (green) or centromeric sequences (red). Regions in white boxes are enlarged to the bottom of the corresponding image for better visualization. Yellow arrows indicate signal-free telomeres; arrowhead indicates chromosome end-to-end fusions. For b and c , 50 metaphases (∼3,360 chromosomes) each of GFP- or TRF2-overexpressing cells were examined for telomeric abnormality. All quantifications were carried out blindly. Each point on the scatter plot represents a single metaphase. Mean values are indicated in red. Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. ( b ) Quantification of signal-free telomeres in HeLa1.2.11 cells overexpressing GFP or TRF2. ( c ) Quantification of chromosome end-to-end fusions in HeLa1.2.11 cells overexpressing GFP or TRF2. ( d ) Schematic diagram of Fusion PCR analysis. ( e ) Individual chromosome end-to-end fusions assessed by Fusion PCR. HeLa1.2.11 cells overexpressing GFP or TRF2 were harvested at PD6. Multiple aliquots of 100 ng of genomic DNA were independently subjected to fusion PCR using a mix of XpYp, 17p and 21q subtelomeric primers. PCR products were resolved on 1% agarose-TBE gel and detected by Southern hybridization with an XpYp-specific subtelomeric probe. ( f ) Representative sequence of fusion molecules between XpYp, 17p and 21q. The fusion points, size of deletion, and microhomology (in red) are indicated.

Article Snippet: Immunoblots were incubated with a mouse monoclonal anti-TRF2 (BD Transduction Laboratories, 1:500), followed by a horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunResearch).

Techniques: Infection, Expressing, Two Tailed Test, Hybridization, Sequencing

Journal: Nature Communications

Article Title: Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions

doi: 10.1038/ncomms10132

Figure Lengend Snippet: ( a ) Formation of thinly stretched telomere bridges between anaphase chromosomes in HeLa1.2.11 cells overexpressing TRF2. Telomeric DNAs were detected by in situ hybridization with a PNA telomeric probe (red). Chromosomes were stained with DAPI (blue). ( b ) Quantification of telomeric anaphase bridges and chromosome end-to-end fusions in HeLa1.2.11 cells overexpressing TRF2 at PD3 and PD7. HeLa1.2.11 cells were infected with lentiviruses expressing TRF2. Parallel cultures were collected at PD3 or PD7 for PNA telomere-FISH to examine telomeric anaphase bridges or for metaphase spreading followed by PNA telomere-FISH to examine chromosome end-to-end fusions. ( c ) Quantification of fragile telomeres in HeLa1.2.11 cells overexpressing GFP control or TRF2 at PD3. Cells were collected for metaphase spreading followed by PNA telomere-FISH to examine fragile telomeres. Representative fragile telomeres are labelled by yellow arrows on images at the left panel. All quantifications were carried out blindly. For b and c , mean values are indicated in red. Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. ( d ) TRF2 overexpression stalled replication at telomeres. Representative chromatin fibre FISH images showed the incorporation of IdU (blue) or CldU (green) at telomeric (red) and adjacent subtelomeric regions in LOX cells infected with lentiviruses expressing luciferase control or TRF2. At PD3, cells in logarithmic growth were labelled sequentially with 30 μM of IdU and then CldU for 4 h each before Chromatin fibre-FISH analysis was carried out. Telomeres were identified by FISH with a telomeric repeat probe. IdU and CldU were identified by immunostaining with analogue-specific antibodies. Dotted line represents the start of telomeric sequences. We did not see dual IdU and CldU labelling at replicating telomeres due to the long labelling time (4 h) used for each halogenated nucleotide. ( e ) Quantification of fraction of telomeric fragments that was labelled with CldU and/or IdU. For control of stalled replication, cells were treated with 1 μg ml −1 aphidicolin for 16 h before they were labelled with IdU and CldU in the presence of aphidicolin (see for representative images).

Article Snippet: Immunoblots were incubated with a mouse monoclonal anti-TRF2 (BD Transduction Laboratories, 1:500), followed by a horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunResearch).

Techniques: In Situ Hybridization, Staining, Infection, Expressing, Two Tailed Test, Over Expression, Luciferase, Immunostaining

Journal: Nature Communications

Article Title: Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions

doi: 10.1038/ncomms10132

Figure Lengend Snippet: ( a ) Representative anaphase images showing the staining of PICH, telomeres, and centromeres in HeLa1.2.11 cells overexpressing TRF2. Cells were infected with lentivirus expressing TRF2 and collected at PD2 after infection for immunostaining-FISH analysis. Telomeres (magenta) and centromeres (red) were identified by PNA FISH. PICH (green) were identified by immunostaining with an anti-PICH antibody. Chromosomes were stained with DAPI (blue). PICH-aligned telomeric anaphase bridges were marked by white arrows. Note that the image represents a single section on the z axis. ( b ) Quantification of PICH bridges, as well as centromere-associated PICH bridges, in HeLa1.2.11 cells overexpressing GFP control or TRF2. Bars represent mean values and s.e.m. (>150 anaphases from three independent experiments examined for each line). Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. ( c ) Quantification of fraction of telomeric UFBs that associate with the PICH protein. 75 telomeric UFB-containing anaphases from three independent experiments were examined for the association between PICH and telomeric UFB. ( d ) Representative anaphase images showing the staining of PICH and centromeres in HeLa1.2.11 cells treated with 20 μM DNA topoisomerase II inhibitor ICRF-159.

Article Snippet: Immunoblots were incubated with a mouse monoclonal anti-TRF2 (BD Transduction Laboratories, 1:500), followed by a horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunResearch).

Techniques: Staining, Infection, Expressing, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions

doi: 10.1038/ncomms10132

Figure Lengend Snippet: ( a ) Long telomere length exacerbates TRF2-induced telomeric UFBs. Left panel: quantification of telomeric UFBs in cells with different mean telomere lengths. Approximately 50 anaphases from each cell line were analysed for telomeric UFBs. Mean values are indicated in red. Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. No telomeric UFBs were detected in HeLa1.2.11 and HT1080 cells infected with lentivirus overexpressing GFP. Telomeres in UM-UC-3 cells (∼3 kb) were pre-extended by expression of the telomerase RNA subunit (hTR) for 11 days (to ∼8 kb) and 20 days (to ∼15 kb). Cells were infected with lentivirus expressing TRF2, and then fixed for telomeric FISH 48 h after infection. TRF2 overexpression did not induce any telomeric UFBs in control UM-UC-3 cells that were infected with an empty lentiviral vector. Right panel: terminal restriction fragment analysis in UM-UC-3 cells expressing vector control or hTR using a telomeric repeat probe. ( b ) TRF2 overexpression fails to induce telomere shortening in cells containing very short telomere length.

Article Snippet: Immunoblots were incubated with a mouse monoclonal anti-TRF2 (BD Transduction Laboratories, 1:500), followed by a horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunResearch).

Techniques: Two Tailed Test, Infection, Expressing, Over Expression, Plasmid Preparation