Journal: STAR Protocols

Article Title: An optimized proximity ligation assay to detect telomere dysfunction induced foci in human and mouse cells

doi: 10.1016/j.xpro.2022.101610

Figure Lengend Snippet: Primary and secondary antibodies for PLA, IF, and IF-Telomere FISH

Article Snippet: Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (against Rabbit TRF2 and 53BP1, 1: 500) PRID: AB_2630356 , Abcam , Cat#ab150077.

Techniques:

Journal: STAR Protocols

Article Title: An optimized proximity ligation assay to detect telomere dysfunction induced foci in human and mouse cells

doi: 10.1016/j.xpro.2022.101610

Figure Lengend Snippet:

Article Snippet: Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (against Rabbit TRF2 and 53BP1, 1: 500) PRID: AB_2630356 , Abcam , Cat#ab150077.

Techniques: In Situ, Fluorescence, Blocking Assay, Neutralization, Hybridization, Cell Culture

Journal: bioRxiv

Article Title: The ERCC6L2-MRI-KU complex coordinates NHEJ at staggered DNA double-strand breaks

doi: 10.1101/2025.11.28.691009

Figure Lengend Snippet: A. c-NHEJ at dysfunctional telomeres with and without overhang. Functional telomeres are formed by a repetitive array of TTAGGG repeats ending with a long (50-300 nt) 3’ overhang bound by the shelterin complex (TRF1, TRF2, RAP1, TIN2, TTP1, POT1). At newly replicated leading end telomeres the overhang is formed by the Exonuclease APOLLO. In the absence of TRF2, telomeres still retaining their overhang are fused by c-NHEJ (top). In the absence of APOLLO and RAP1, the blunt telomeres resulting from replication cannot be resected and are fused by c-NHEJ (bottom). B. Immunoblot for TRF2 deletion, CHK2 phosphorylation as indication of ATM activation and actin as loading control in Ercc6l2 +/+ and Ercc6l2 -/- MEFs 108 h after transduction with single guide RNA (sgRNA) targeting Trf2 (sgTrf2). C. Representative FISH of metaphase spreads of Ercc6l2 positive and negative 108 h after Trf2 deletion. Telomeres were detected with Cy3-(TTAGGG)3 (green) and DNA was stained with DAPI (magenta). Green arrows highlight chromosome-type fusions. D. Quantification of telomeres involved in chromosome fusions per metaphase after deletion of Trf2 as in (B-C). Data from 3 independent experiments, 10 metaphases per experiment (n = 30 total), with median. E. Immunoblot for Rap1 deletion, CHK2 and actin in Ercc6l2 +/+ and Ercc6l2 -/- MEFs 120 h after transduction with single guide RNA (sgRNA) targeting Rap1 (sgRAp1) and/or Apollo (sgApollo). F. Representative FISH of metaphase spreads of Ercc6l2 positive and negative MEFs 120 h after Apollo and Rap1 deletion. Telomeres were detected with Cy3-(TTAGGG)3 (green) and DNA was stained with DAPI (magenta). Green arrows highlight chromosome-type fusions. White arrows highlight chromatid-type fusions. G. Quantification of telomeres involved in chromosome fusions per metaphase after deletion of Rap1 and Apollo as in (e-f). Data from 2 (no sgRNAs) or 3 (sgRap1 + sgApollo) independent experiments, 10 metaphases per experiment with median. Ordinary one-way analysis of variance (ANOVA). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; ns, not significant.

Article Snippet: Western blot was performed with 5% milk in PBS containing 0.1% (v/v) Tween-20 (PBS-T) using the following antibodies: β-actin (#3700; Cell Signaling), RAP1 (#5433, Cell Signalling) and TRF2 (#13136, Cell Signaling), followed by goat anti-rabbit (31460, Invitrogen) or anti-mouse (31430, Invitrogen) IgG–HRP peroxidase secondary antibody.

Techniques: Functional Assay, Western Blot, Phospho-proteomics, Activation Assay, Control, Transduction, Staining

Journal: bioRxiv

Article Title: The ERCC6L2-MRI-KU complex coordinates NHEJ at staggered DNA double-strand breaks

doi: 10.1101/2025.11.28.691009

Figure Lengend Snippet: Depletion of the TRF2 shelterin component exposes DSBs with extended C-strand 3′ overhangs (top left box), while excision and nucleolytic processing of uracil residues (red asterisks) generated by AID-dependent cytosine deamination within IgH switch regions result in staggered DSBs of variable overhang length and polarity (bottom left box). In both scenarios, DSBs bearing long 3′ overhangs trigger 53BP1-RIF1-dependent recruitment of shieldin, which co-recruits CST to stimulate 5′-3′ fill-in synthesis, either from DNA termini or from internal positions, thereby generating DSBs with blunt or short-recessed ends. Shieldin-CST-Polα-primase-mediated conversion of blunt DSBs produces ends that are directly ligatable by the short-range c-NHEJ synapsis complex (top right box). By contrast, internally primed fill-in synthesis at telomeric DSBs, or the generation of short-recessed DNA ends by AID, produces intermediates that require ERCC6L2-MRI-dependent coordination of KU at DNA ends (bottom right box) for additional end processing or nucleases, and/or DNA synapsis.

Article Snippet: Western blot was performed with 5% milk in PBS containing 0.1% (v/v) Tween-20 (PBS-T) using the following antibodies: β-actin (#3700; Cell Signaling), RAP1 (#5433, Cell Signalling) and TRF2 (#13136, Cell Signaling), followed by goat anti-rabbit (31460, Invitrogen) or anti-mouse (31430, Invitrogen) IgG–HRP peroxidase secondary antibody.

Techniques: Generated

Journal: Molecular and Cellular Biology

Article Title: p53 Differentially Inhibits Cell Growth Depending on the Mechanism of Telomere Maintenance

doi: 10.1128/mcb.24.13.5967-5977.2004

Figure Lengend Snippet: FIG. 1. Expression of ts-p53TA inhibits DNA replication in ALT cell lines. (A) HIO107 cells transiently transfected with ts-p53TA were cultured at either 39 or 32°C for 48 h, pulsed for 40 min with BrdU, and then harvested to detect p53 (red) and BrdU (green). DNA was detected with DAPI. Note that the ts-p53TA-positive cells cultured at 32°C have not incorporated BrdU. Due to overexpression of p53, the protein may be present in both the nucleus and the cytoplasm. (B) Quantitation of data such as those shown in panel A generated from the indicated cell lines. The ALT cell lines expressing ts-p53TA have a reduced frequency of BrdU-positive cells when cultured at 32°C compared to the frequency of BrdU-positive cells transfected with empty vector. (C) FACS analysis of HIO107 cells cotransfected with the CD19 cell surface marker and ts-p53TA. Cells were cultured and pulsed with BrdU as in panel A and then stained to detect BrdU (y axis) and CD19 (x axis). (D) Quantitation of data such as those shown in panel C for the indicated cell lines. The percentage of BrdU-positive cells in the transfected population is determined by gating on the CD19-positive population. The expression of ts-p53TA does not inhibit BrdU incorporation at 32°C in the telomerase-positive cell lines relative to cells transfected with empty vector in either assay.

Article Snippet: The precleared chromatin was incubated overnight at 4°C with the following primary antibodies: goat polyclonal human TRF2 (Imgenex), goat polyclonal p53 (C-19; Santa Cruz), or goat serum IgG (Santa Cruz).

Techniques: Expressing, Transfection, Cell Culture, Over Expression, Quantitation Assay, Generated, Plasmid Preparation, Marker, Staining, BrdU Incorporation Assay

Journal: Molecular and Cellular Biology

Article Title: p53 Differentially Inhibits Cell Growth Depending on the Mechanism of Telomere Maintenance

doi: 10.1128/mcb.24.13.5967-5977.2004

Figure Lengend Snippet: FIG. 2. Characterization of ALT-positive HIO107 or telomerase-positive HIO114 cell lines expressing either wild-type (WT) or transactivation- incompetent (TA) p53. (A) Western blot analysis of whole-cell extracts from the indicated cell lines prepared after 48 h of growth at 39 or 32°C probed to detect p53, p21WAF1, and T-Ag as a loading control. p53-TA does not result in accumulation of p21WAF1 protein. P, parental cell line. (B) Frequency of apoptotic cells, detected by 7 AAD and annexin V staining, in the indicated cell lines. Results are shown as the increase (n-fold) in apoptosis when cells are cultured at 32°C compared to the level of background apoptosis in the culture at 39°C. Relative to ts-p53WT, the ts-p53TA allele is impaired in its ability to induce apoptosis at the permissive temperature of 32°C. (C) Growth curves of the HIO107 ALT (squares) or HIO114 telomerase-positive (circles) derived cell lines at 39°C (open symbols; solid line) or 32°C (filled symbols; dashed line). Expression of p53-TA causes growth inhibition in the HIO107 ALT background. The results are the summary of experiments done in triplicate. Where they are not visible, the standard error bars are below the resolution of the graph.

Article Snippet: The precleared chromatin was incubated overnight at 4°C with the following primary antibodies: goat polyclonal human TRF2 (Imgenex), goat polyclonal p53 (C-19; Santa Cruz), or goat serum IgG (Santa Cruz).

Techniques: Expressing, Western Blot, Control, Staining, Cell Culture, Derivative Assay, Inhibition

Journal: Molecular and Cellular Biology

Article Title: p53 Differentially Inhibits Cell Growth Depending on the Mechanism of Telomere Maintenance

doi: 10.1128/mcb.24.13.5967-5977.2004

Figure Lengend Snippet: FIG. 3. Expression of ts-p53TA perturbs APB frequency and causes an S phase delay in HIO107 ALT cells. (A) The frequency of APB-positive cells is increased two- to threefold when cell lines expressing either ts-p53WT or ts-p53TA are cultured at 32°C. APBs were detected by costaining the cells with a goat polyclonal antibody against PML and a rabbit polyclonal antibody (FC-08) against TRF2. (B) The HIO107-TA ALT cell line incorporates BrdU, but the cells accumulate in early S phase after 72 h at 32°C, while the HIO114 cell line is unaffected by growth at 32°C. The left panel shows a FACS analysis of BrdU intensity (y axis) versus DNA content (x axis). The right panel shows the cell cycle profiles generated from the data on the left of the figure (y axis, number of events; x axis, DNA content). (C) The parental HIO107 and the HIO107-TA cell lines were arrested by exposure to HU and, following release, cultured at either 32 or 39°C for the indicated time prior to harvest. The presence of active ts-p53TA prevents cells from progressing through S phase following the removal of HU. y axis, number of events; x axis, DNA content. (D) Quantitation of the cell cycle distribution of the populations shown in panel C. HIO107-TA cells accumulate in S phase for up to 24 h, suggesting that S phase is delayed when ts-p53TA is expressed. (E) Quantitation of the pattern of BrdU incorporation in HIO107-TA cells after 72 h of growth at 32°C. The majority of the cells are BrdU positive at 39°C, while at 32°C many of the cells are either BrdU negative or have punctate BrdU staining. (F) At 32°C, some HIO107-TA cells exhibit punctate BrdU staining which colocalizes with the telomeric protein TRF2 (arrow). The other two staining patterns also shown are diffuse nuclear staining or no BrdU incorporation.

Article Snippet: The precleared chromatin was incubated overnight at 4°C with the following primary antibodies: goat polyclonal human TRF2 (Imgenex), goat polyclonal p53 (C-19; Santa Cruz), or goat serum IgG (Santa Cruz).

Techniques: Expressing, Cell Culture, Generated, Quantitation Assay, BrdU Incorporation Assay, BrdU Staining, Staining

Journal: Molecular and Cellular Biology

Article Title: p53 Differentially Inhibits Cell Growth Depending on the Mechanism of Telomere Maintenance

doi: 10.1128/mcb.24.13.5967-5977.2004

Figure Lengend Snippet: FIG. 4. ChIP analysis. (A) Telomeric DNA is precipitated by anti- bodies against the telomeric binding proteins TRF2 but not by non- specific goat IgG, irrespective of culture conditions. Immunoprecipi- tation requires formaldehyde cross-linking of proteins to DNA. p53 is also associated with telomeric DNA in the HIO107-TA cell line but only at the permissive temperature of 32°C. Antibodies against TRF2 do not immunoprecipitate centromeric -satellite sequences. (B) Quantitation of the data shown in panel A. Percent input precip- itated (ppted) [(signal strength of telomeric DNA precipitated with the indicated specific antibody background signal obtained with nonspecific IgG)/input signal] 100. (C) Agarose gel of PCR products using primers to amplify the p53 binding site at position 1.4 kb within the p21WAF1 promoter. The template DNAs are identical to those interrogated for the experiment shown in panel C. The predicted product of 113 bp is only detected in DNA from the ChIP assay carried out with an antibody against p53 at 32°C. MW, PhiX HaeIII markers. Antibodies used for all panels are as follows: Gt TRF2, goat polyclonal antibody against TRF2 (Imgenex); Gt p53, goat polyclonal antibody against the carboxy terminus of human p53 (C-19; Santa Cruz); Gt IgG, goat IgG (Santa Cruz).

Article Snippet: The precleared chromatin was incubated overnight at 4°C with the following primary antibodies: goat polyclonal human TRF2 (Imgenex), goat polyclonal p53 (C-19; Santa Cruz), or goat serum IgG (Santa Cruz).

Techniques: Binding Assay, Quantitation Assay, Agarose Gel Electrophoresis

Journal: Molecular and Cellular Biology

Article Title: p53 Differentially Inhibits Cell Growth Depending on the Mechanism of Telomere Maintenance

doi: 10.1128/mcb.24.13.5967-5977.2004

Figure Lengend Snippet: FIG. 5. Expression of p53 mutant alleles that compromise the abil- ity of p53 to suppress recombination does not inhibit DNA replication in ALT cell lines. (A) Immunofluorescent staining of the indicated p53 compound mutant protein (top). DNA was stained with DAPI. West- ern analysis of each p53 compound mutant allele is shown (bottom). (B) Ratio of BrdU incorporation in transfected cells at 39 versus 32°C for the HIO107 and WI38-VA13/2RA ALT-positive and HIO114 tel- omerase-positive cell lines transfected with empty vector or the indi- cated p53 allele. All ratios were normalized to the frequency of BrdU incorporation at the two temperatures in control cells transfected with empty vector in parallel.

Article Snippet: The precleared chromatin was incubated overnight at 4°C with the following primary antibodies: goat polyclonal human TRF2 (Imgenex), goat polyclonal p53 (C-19; Santa Cruz), or goat serum IgG (Santa Cruz).

Techniques: Expressing, Mutagenesis, Staining, BrdU Incorporation Assay, Transfection, Plasmid Preparation, Control