Journal: Molecular and Cellular Biology

Article Title: TIN2-Tethered TPP1 Recruits Human Telomerase to Telomeres In Vivo

doi: 10.1128/mcb.00240-10

Figure Lengend Snippet: FIG. 6. The TPP1 OB-fold is required to rescue telomerase recruitment to telomeres. An shRNA-resistant form of TPP1 is able to restore hTR localization to telomeres in TPP1-depleted cells. However, an shRNA-resistant form of TPP1 lacking the OB-fold cannot restore localization. (A) Parental and TPP1-depleted super-telomerase HeLa cells were subjected to FISH and IF to detect hTR (red), coilin (blue), and TRF2 (green). Merge panels show superimposition of hTR, coilin, and TRF2. Next, parental cells were cotransfected with shTPP1 and either TPP1* or TPP1OB*. Treated cells were subjected to FISH and IF to detect hTR (red), FLAG (blue), and RAP1 (telomere marker, green). Merge panels show superimposition of hTR, FLAG, and RAP1. (B) Plot of the average number of telomere-associated hTR foci per cell in the parental cells and each experimental group. Error bars indicate standard errors calculated with N equal to the number of samples quantitated.

Article Snippet: Next, cells were incubated with one of several combinations of the following primary antibodies at the indicated dilution for 1 h at room temperature: mouse anti-p80 coilin (1:5,000, ) (1), mouse anti-TRF2 (1:1,000; Imgenex Corp., San Diego, CA), rabbit anti-hTERT (1:400; Rockland, Gilbertsville, PA), mouse anti-FLAG (1:500; Sigma-Aldrich, St. Louis, MO), rabbit anti-RAP1(1: 2,000; Novus Biologicals, Littleton, CO), and rabbit anti-53BP1(1:500; Bethyl, Montgomery, TX).

Techniques: shRNA, Marker

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Optogenetic recruitment of talin to the plasma membrane of endothelial cells leads to activation of Rap1. (A) Schematic representation of the optogenetic constructs CIBN–GFP–CAAX and CRY2–mCherry–talin expressed in immortalized mouse lung endothelial cells. The CIBN moiety is anchored to the plasma membrane and recruits CRY2–mCherry–talin upon exposure of the cells to 450 nm (blue) light. Previous work has shown that such recruitment leads to activation of integrin αVβ3 . Rap1 activation, the transition from Rap1–GDP to Rap1–GTP, can also be monitored during this process. (B) Time course of Rap1 activation in endothelial cells in response to blue light illumination. Rap1–GTP was selectively pulled down using agarose beads loaded with the Rap-binding domain of RalGDS and detected using an anti-Rap1 antibody. Upper panel: representative western blots of the Rap1–GTP pull-down assay and total Rap1 in whole-cell lysates (input: 5%). Lower panel: quantitative analysis of Rap1–GTP. The ratio of Rap1–GTP to total Rap1 was calculated and normalized to that observed at time zero when the cells were maintained in the dark. Data represent means±s.e.m. of four experiments (* P <0.05; paired two-tailed Student's t -test). (C) Time course of Rap1 activation in response to blue light in A5 CHO cells stably expressing integrin αIIbβ3, CIBN–GFP–CAAX and CRY2–mCherry–talin. Data represent means±s.e.m. of four experiments (* P <0.05; ** P <0.01; paired two-tailed Student's t -test). (D) Recruitment to the plasma membrane of a CRY2–mCherry–talin mutant (R118E) that cannot interact with Rap1 fails to activate Rap1 in A5 CHO cells. Data represent means±s.e.m. of four experiments (N.S., not significant; ** P <0.01; paired two-tailed Student's t -test). (E) Expression of a CRY2–mCherry–talin mutant (L325R) defective in activating integrins still enables Rap1 activation in these cells upon recruitment of CRY2–mCherry–talin to the plasma membrane in A5 CHO cells. Data represent means±s.e.m. of four experiments (* P <0.05; paired two-tailed Student's t -test).

Article Snippet: The mouse monoclonal antibody to Rap1 was from Proteintech (Rosemont, IL, USA; 67174).

Techniques: Clinical Proteomics, Membrane, Activation Assay, Construct, Binding Assay, Western Blot, Pull Down Assay, Two Tailed Test, Stable Transfection, Expressing, Mutagenesis

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Optogenetic recruitment of talin to the plasma membrane promotes active Rap1 localization to cell edges. (A) Optogenetic recruitment of talin to the plasma membrane promotes active Rap1 localization to the cell periphery in suspended cells. Immortalized murine endothelial cells in suspension expressing CIBN–GFP–CAAX and CRY2–mCherry–talin were illuminated using blue light for 30 min before Rap1–GTP was detected in situ as described in the Materials and Methods. Samples without GST–RalGDS were used as a control (column 1). White arrows indicate enrichment of active Rap1 and CRY2–mCherry–talin at the cell periphery, the former only in response to blue light. Scale bar: 10 µm. (B) Optogenetic recruitment of talin to the plasma membrane enriches active Rap1 localization at cell protrusions in adherent cells. Immortalized murine endothelial cells expressing CIBN–GFP–CAAX and CRY2–mCherry–talin were plated on fibronectin-coated coverslips for 30 min before being illuminated with blue light or maintained in the dark for 30 min. Samples were fixed and in situ Rap1–GTP assay was performed as described in the Materials and Methods. Samples without GST–RalGDS incubation were used as a negative control and are shown in column 1. In these representative images, cell protrusions are highlighted by the small box in the main panel and presented as magnified insets on the bottom right of each image. Blue light illumination induces active Rap1 localization on cell lamellipodium-like protrusions (column 4) and pseudopodium-like protrusions (column 5). Note that such signal enrichment was not seen in cells maintained in the dark (columns 2 and 3) despite the formation of cell protrusions. Scale bars: 20 µm (main panel); 1 µm (inset). Images in A,B are representative of three independent experiments.

Article Snippet: The mouse monoclonal antibody to Rap1 was from Proteintech (Rosemont, IL, USA; 67174).

Techniques: Clinical Proteomics, Membrane, Suspension, Expressing, In Situ, Control, Incubation, Negative Control

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Overexpression of SHANK3 blocks Rap1 activation induced by talin recruitment to the plasma membrane. (A–C) SHANK3 tagged with Myc–mAzurite was transfected into A5 CHO cells stably expressing CIBN–GFP–CAAX and CRY2–mCherry–talin. Cells triple positive for mAzurite, GFP and mCherry were sorted by flow cytometry. Cells expressing Myc–mAzurite without SHANK3 served as a control. (A) Western blot analysis of SHANK3–Myc–mAzurite expression in these cells. β-actin served as a loading control. SHANK3 overexpression did not affect the levels of CRY2–mCherry–talin. The images represent two independent experiments. (B) SHANK3 overexpression inhibits Rap1 activation in response to the optogenetic recruitment of talin to the plasma membrane. Data represent means±s.e.m. of five experiments (N.S., not significant; ** P <0.01; paired two-tailed Student's t -test). (C) SHANK3 blunts activation of integrin αIIbβ3 in response to the optogenetic recruitment of talin to the plasma membrane. Activation of integrin αIIbβ3 was monitored by flow cytometry using the PAC-1 antibody and expressed as the fold increase relative to that observed when cells were maintained in the dark. Data represent means±s.e.m. of five experiments (* P <0.05; paired two-tailed Student's t -test). (D–G) Lentiviruses encoding the WT SPN domain of SHANK3 [mAzurite–FLAG–SPN (WT)], the R12C SPN mutant [mAzurite–FLAG–SPN (R12C)] or the L68P SPN mutant [Myc–mAzurite–FLAG–SPN (L68P)] were transduced into immortalized murine lung endothelial cells expressing CIBN–GFP–CAAX and CRY2–mCherry–talin. Cells infected with empty lentiviral vector served as controls. (D) WT SPN, but not R12C or L68P SPN, inhibits Rap1 activation following optogenetic recruitment of talin to the plasma membrane. Data represent mean±s.e.m. of four experiments (N.S., not significant; * P <0.05; paired two-tailed Student's t -test). (E) WT SPN, but not the R12C or L68P SPN mutants, inhibits specific fibrinogen binding to integrin αVβ3 upon optogenetic recruitment of talin to the plasma membrane. Data represent means±s.e.m. of eight experiments (N.S., not significant; * P <0.05; ** P <0.01; paired two-tailed Student's t -test). (F,G) Duolink proximity ligation assay (PLA) was performed to examine the effects of SHANK3 SPN on the association of Rap1 with CRY2–mCherry–talin in endothelial cells. (F) Schematic representation of the Duolink PLA. Created in BioRender by Liao, Z., 2025. https://BioRender.com/m47c469 . This figure was sublicensed under CC-BY 4.0 terms. (G) After 30 min of incubation at room temperature in the absence or presence of blue light illumination, cells were fixed, permeabilized and stained with rabbit anti-mCherry and mouse anti-Rap1 antibodies. Then, Duolink PLA flow cytometry was performed to assess the interaction between CRY2–mCherry–talin and Rap1. Cells kept in the dark and untreated with primary antibodies served as controls. Data represent means±s.e.m. of four experiments (* P <0.05; paired two-tailed Student's t -test).

Article Snippet: The mouse monoclonal antibody to Rap1 was from Proteintech (Rosemont, IL, USA; 67174).

Techniques: Over Expression, Activation Assay, Clinical Proteomics, Membrane, Transfection, Stable Transfection, Expressing, Flow Cytometry, Control, Western Blot, Two Tailed Test, Mutagenesis, Infection, Plasmid Preparation, Binding Assay, Proximity Ligation Assay, Incubation, Staining

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Optogenetic recruitment of talin to the plasma membrane impairs Rap1 interaction with SHANK3. (A) Schematic representation of the Duolink proximity ligation assay (PLA). Cells were fixed, permeabilized and stained with rabbit anti-SHANK3 and mouse anti-Rap1 antibodies before Duolink PLA was performed to assess the proximity of SHANK3 to Rap1. Created in BioRender by Liao, Z., 2025. https://BioRender.com/b40n281 . This figure was sublicensed under CC-BY 4.0 terms. (B) Immortalized murine lung endothelial cells expressing CRY2–mCherry–talin and CIBN–GFP–CAAX were plated on fibrinogen overnight, fixed, permeabilized and stained with anti-SHANK3 and anti-Rap1 antibodies. PLA was performed to evaluate colocalization of endogenous SHANK3 and Rap1. Cell nuclei were counterstained with DAPI and cells were imaged by confocal microscopy. Cells kept in the dark and untreated with primary antibodies served as controls. Two areas within the images with merged signals for PLA and CIBN–GFP–CAAX are highlighted with boxes and presented as magnified insets on the bottom. PLA signals were observed within the cytoplasm (inset on the left) and on the plasma membrane (inset on the right). Scale bars: 35 µm (main panel); 10 µm (inset). Images represent two independent experiments. (C) Immortalized murine lung endothelial cells in suspension were either kept in the dark or exposed to blue light illumination for the indicated times, before being subjected to Duolink PLA assay and analyzed by flow cytometry to quantitatively assess the interaction of endogenous SHANK3 and Rap1. Cells kept in the dark and untreated with primary antibodies served as controls. Data represent means±s.e.m. of five experiments (N.S., not significant; * P <0.05; paired two-tailed Student's t -test). Cells transduced with lentivirus encoding shRNA to knock down SHANK3 were also used as a further control to demonstrate the specificity of the PLA signal in four out of the five experiments (* P <0.05; unpaired two-tailed Student's t -test).

Article Snippet: The mouse monoclonal antibody to Rap1 was from Proteintech (Rosemont, IL, USA; 67174).

Techniques: Clinical Proteomics, Membrane, Proximity Ligation Assay, Staining, Expressing, Confocal Microscopy, Suspension, Flow Cytometry, Two Tailed Test, Transduction, shRNA, Knockdown, Control

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Detection of TERRA and hTR by smiFISH in HeLa cells. The percentage of cells, in which at least 1 TERRA-hTR colocalization event is detected, is indicated (mean ± SD; n = 5; 298 cells analyzed). Scale bar, 5 μm. ( B ) Quantification of the number of TERRA-hTR colocalizations per nucleus (mean ± SD; n = 5; 298 cells analyzed). ( C ) Detection of TERRA, hTR, and telomeres by smiFISH/RAP1 IF in HeLa cells (mean ± SD; n = 2; 102 cells analyzed). Scale bar, 5 μm. ( D ) Quantification of the number of TERRA-hTR colocalizations per cell detected at telomeres (TERRA-hTR-RAP1) and outside telomeres (TERRA-hTR w/o RAP1) (mean ± SD; n = 2; 102 cells analyzed). ( E ) Number of hTR-RAP1 colocalizations per cell with and w/o TERRA (mean ± SD; n = 2; 102 cells analyzed). ( F ) Quantification of the number of TERRA-hTR foci at telomeres (TERRA-hTR-RAP1) and outside telomeres (TERRA-hTR w/o RAP1) during G 1 , S, and G 2 phase in HeLa cells upon cell synchronization (mean ± SD; n = 2; total number of cells analyzed: 54 (G 1 phase), 46 (S phase), and 45 (G 2 phase). Fraction of hTR-TERRA foci at telomeres: 20.9 ± 3.3% in G 1 -phase cells, 40.2 ± 11% in S-phase cells, and 41.1 ± 5.5% in G 2 -phase cells.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques:

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Telomere length measurement by TRF through Southern blot in HeLa cells in the indicated conditions. NT, untreated; CTR, DMSO-treated. Estimated average telomere length and elongation rate for each sample are indicated in the table below. Average ± SD from two technical replicates. ( B ) Detection of TERRA and telomeres by smiFISH/IF in CTR, 7PD rescue, and 24PD rescue cells. Scale bar, 5 μm. ( C ) Quantification of the number of telomeric TERRA foci detected per nucleus by smiFISH/IF (each dot represents a nucleus) (mean ± SD; n = 2; 124 CTR cells, 111 7PD rescue cells, and 112 24PD rescue cells analyzed). Unpaired nonparametric Kruskal-Wallis test coupled with post hoc Dunn’s multiple-comparison test: ** P < 0.01, *** P < 0.001. ( D and E ) Distribution analyses of the number of telomeric TERRA foci per cell. Two-way analysis of variance (ANOVA) test: ** P < 0.01. Post hoc Tukey’s multiple-comparison test: group 1 to 4 P values = 0.07 (CTR versus 7PD rescue), 0.04 (7PD rescue versus 24PD rescue), not significant (ns) (CTR versus 24PD rescue). ( F ) Detection of TERRA, hTR, and telomeres by smiFISH/IF in CTR, 7PD rescue, and 24PD rescue cells. Scale bar, 5 μm. ( G ) Quantification of the percentage of TERRA-hTR foci colocalizing at telomeres and extratelomeric (mean ± SD; n = 2; 124 CTR cells, 111 7PDs rescue cells, and 112 24PDs rescue cells analyzed). Two-way ANOVA test: **** P < 0.0001. ( H ) Distribution analyses of the number of TERRA-hTR-RAP1 colocalizing foci per nucleus. Two-way ANOVA test: **** P < 0.0001; multiple-comparison test: P = 0.0003 for 7PD versus 24PD rescue, P < 0.0001 for CTR versus 7PD rescue, P = 0.001 CTR versus 24PD rescue. ( I ) Number of telomeric hTR foci not colocalizing with TERRA per cell detected by smiFISH/IF. Kruskal-Wallis test: P = 0.0025.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Southern Blot, Comparison

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Detection of TERRA and RAP1 by smiFISH/IF in HeLa cells. An example of a colocalization event between TERRA and RAP1 is shown in the image magnifications. DAPI is used to stain nuclei. Scale bar, 5 μm. ( B ) Integrated density quantification of RAP1 foci colocalizing and not colocalizing with TERRA foci in HeLa cells as detected by smiFISH/IF. Each dot represents a single RAP1 focus. Mean ± SD is shown. A total of 110 cells were analyzed in three independent biological replicates. Statistical significance was assessed by Mann-Whitney test. **** P < 0.0001. ( C ) Integrated density quantification of RAP1 foci colocalizing and not colocalizing with TERRA foci in the indicated samples. Each dot represents a single RAP1 focus. Mean ± SD is shown from the following number of samples and biological replicates: 64 CTR cells ( n = 2), 67 BIBR1532 cells (123PDs of treatment with BIBR 1532) ( n = 2), 111 7PD rescue cells ( n = 2), 151 POT1 WT cells ( n = 3), and 148 POT1-ΔOB cells ( n = 3). The Mann-Whitney test was used to assess statistical significance. **** P < 0.0001.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Staining, MANN-WHITNEY

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Detection of TERRA and telomeres in HeLa cells by smiFISH/IF. Image acquisitions were performed using three-dimensional structured illumination microscopy (3D-SIM). Examples of TERRA foci colocalizing with a single telomere (top images) or telomere doublet (bottom images) are displayed. TERRA is shown in red; telomeres are in green. Scale bar is indicated in the rotated view images. ( B ) Quantification of the fraction of single telomeres versus telomere doublets colocalizing with TERRA. Data are shown as percentage of TERRA-colocalizing telomeres and represent mean ± SD from three independent biological replicates for a total of 30 cells and 663 TERRA-colocalizing RAP1 foci analyzed. ( C and D ) Quantification of the integrated density (C) and volume (D) of RAP1 foci colocalizing (with TERRA) and not colocalizing (without TERRA) with TERRA. Both TERRA-single telomere and TERRA-telomere doublet colocalizations were considered. Data are shown as arbitrary units (a.u.), in (C), and μm 3 , in (D), and represents mean ± SD from three independent biological replicates for a total of 30 cells and 663 TERRA-colocalizing RAP1 foci analyzed. The Mann-Whitney test was used to assess statistical significance. **** P < 0.0001. ( E ) Quantification of the integrated density of all TRF1-mCherry foci and TRF1-mCherry foci colocalizing with MS2-tagged telomere 15q TERRA transcripts per nucleus. Forty nuclei corresponding to 3690 telomeres and 73 telomeres colocalizing with MS2-TERRA transcripts were analyzed from imaging datasets obtained in . Statistical analysis was performed with a Kolmogorov-Smirnov test: P ≤ 0.0001.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Microscopy, MANN-WHITNEY, Imaging

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Northern blot analysis of TERRA in HeLa cells upon TERRA-ASO or control ASO (ASO SCR) transfection. Bottom image shows 18 S rRNA band upon gel run. ( B ) Quantification of TERRA signal from Northern blot analyses of TERRA-ASO–transfected cells shown as fold over ASO SCR (dashed line). * P < 0.05; mean ± SD, n = 2. ( C ) RT-qPCR analyses of TERRA expression from the indicated telomeres in TERRA-ASO cells shown as fold over ASO SCR. Mean ± SD from four independent biological replicates. Unpaired t test: ** P < 0.01, *** P < 0.001. ( D ) Integrated density quantification of TERRA foci colocalizing with RAP1 foci. Each dot represents a single TERRA signal (mean ± SD; n = 2; 131 ASO SCR cells and 122 TERRA-ASO cells analyzed). Mann-Whitney test: ** P < 0.01. ( E ) Quantification of the number of hTR foci detected per nucleus in TERRA-ASO and ASO SCR cells (mean ± SD; n = 2; 131 ASO SCR cells and 122 TERRA-ASO cells analyzed). Mann-Whitney test: **** P < 0.0001. ( F ) RT-qPCR quantification of hTR levels using primer pairs detecting the precursor or mature RNA ( , ). Results are shown as fold change over ASO SCR (dashed line) (mean ± SD, n = 2). U6 gene was used for normalization . ( G ) Detection of hTR and telomeres by smiFISH/IF. Scale bar, 5 μm. ( H ) Quantification of the number of telomeric hTR foci detected per nucleus. Data are shown as number of RAP1-hTR colocalizations per cell (each dot represents a cell) (mean ± SD, n = 2; 131 ASO SCR cells and 122 TERRA-ASO cells analyzed). Mann-Whitney test: **** P < 0.0001. ( I and J ) Distribution analysis of the number of RAP1-hTR colocalizations detected per cell. Two-way ANOVA test: ** P < 0.01.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Northern Blot, Control, Transfection, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: EMBO Reports

Article Title: Five dysfunctional telomeres predict onset of senescence in human cells

doi: 10.1038/embor.2011.227

Figure Lengend Snippet: Relationship between telomere length and meta-TIFs. ( A ) Percentage of meta-TIFs with detectable (+) or undetectable (−) telomere FISH signals (mean±s.d., n =3). ( B ) Comparison of relative fluorescence intensity of γ-H2AX IF (red) and telomere FISH (green). ( C ) Quantification of relative telomeric fluorescence intensity between the γ-H2AX-positive and -negative sister telomeres of a chromosome-type meta-TIF (mean±s.d., n =3). ( D ) Cyto-centrifuged chromosomes stained with DAPI (blue), γ-H2AX IF (red) and TRF2 or RAP1 IF (green). DAPI, 4′,6-diamidino-2-phenylindole; DDR, DNA damage response; FISH, fluorescence in situ hybridization; γ-H2AX, phosphorylated H2AX; IF, immunofluorescence; meta-TIF, metaphase telomere dysfunction-induced focus; PD, population doubling; Tel, telomere.

Article Snippet: The primary antibodies used were γ-H2AX (613402, BioLegend, or IHC-00059-1, Bethyl Laboratories), MRE11 (100-142 G2, Novus Biologicals), NBS1 (611871, BD Transduction), Chk2-Thr 68 (2661S, Cell Signaling Technologies), TRF2 (110-57130, Novus Biologicals) and RAP1 (NS100-292, Novus Biologicals).

Techniques: Comparison, Fluorescence, Staining, In Situ Hybridization, Immunofluorescence