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