Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: p300 acetylates TRF2 in vivo and the two proteins form a stable complex. ( A ) Endogenous TRF2 is acetylated by p300. HEK293 cells were transfected with p300-HA or Flag-PCAF and subjected to immunoprecipitation with anti-TRF2 or normal IgG antibodies, followed by immunoblotting with anti-acetylated lysine antibody (Ac-Lys). ( B ) HEK293 cells were co-transfected with Flag-TRF2 or Flag-p53 and either p300-HA or p300ΔHAT-HA and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblotting with anti-acetylated lysine antibody. The arrowheads mark the positions of p300-HA and p300ΔHAT-HA. ( C ) HEK293 cells were co-transfected with Flag-TRF2 or Flag-p53 and either p300-HA or p300ΔHAT-HA and subjected to immunoprecipitation with anti-HA antibody, followed by immunoblotting with anti-Flag antibody. ( D ) HEK293 cells were transfected with Flag-TRF2 or Flag-TRF1 and subjected to immunoprecipitation with anti-TRF2, anti-TRF1 or anti-Flag antibodies, followed by immunoblotting with anti-p300 antibody. ( E ) HEK293 cells were subjected to immunoprecipitation with either anti-TRF2 or anti-TRF1 antibodies, followed by immunoblotting with anti-p300 antibody. IgG antibody was used as a negative control. ( F ) HEK293 cells were analysed by indirect immunofluorescence for co-localization of TRF2 with p300. Immunofluorescence was used to detect endogenous TRF2 (red) and p300 (green). DNA was stained with DAPI (blue).

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: In Vivo, Transfection, Immunoprecipitation, Western Blot, Negative Control, Immunofluorescence, Staining

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: Identification of the interaction domain of TRF2 with p300. ( A ) Schematic representation of TRF2 and its deletion variants used in this study. ( B ) HEK293 cells were co-transfected with p300-HA and various TRF2-Myc and subjected to immunoprecipitation with anti-HA antibody, followed by immunoblotting with anti-Myc antibody. The TRF2-Myc proteins were detected by immunoblotting with anti-Myc antibody. ( C ) Schematic representation of TRF2 and its deletion variants (GAR, TRFH, Hinge and Myb). ( D ) The various GST-TRF2 proteins were affinity-purified and incubated with lysates prepared from cells expressing p300-HA, followed by detecting p300-HA. The purified GST fusion proteins were visualized by Coomassie staining. The arrowhead indicates the degradation product of GST-Hinge. Molecular mass makers are shown in kilodaltons.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Transfection, Immunoprecipitation, Western Blot, Affinity Purification, Incubation, Expressing, Purification, Staining

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: Identification of the acetylation site on TRF2. ( A ) HEK293 cells were co-transfected with p300-HA and various TRF2-Myc and subjected to immunoprecipitation with anti-Myc antibody, followed by immunoblotting with anti-acetylated lysine antibody. The various TRF2-Myc proteins were detected by immunoblotting with anti-Myc antibody. ( B ) The in vitro TRF2 acetylation assay was carried out using the various GST-TRF2 proteins. The acetylated proteins were detected by immunoblotting with anti-acetylated lysine antibody. The purified GST fusion proteins were visualized by Coomassie staining. Molecular mass makers are shown in kilodaltons. The arrowheads indicate the degradation product of GST-Hinge. ( C ) The various GST-TRF2 proteins were subjected to the in vitro acetylation assay using GST-p300 in the presence of [ 14 C] acetyl coenzyme A. The acetylated proteins were detected by autoradiography. The purified GST fusion proteins were visualized by Coomassie staining. Molecular mass makers are shown in kilodaltons. The arrowheads indicate the degradation product of GST-Hinge. ( D ) MS/MS spectra corresponding to the acetylated peptide (AAFKacTLSGAQDSEAAFAK) from TRF2. HEK293 cells were co-transfected with Flag-TRF2 and p300-HA and subjected to immunoprecipitation with anti-Flag antibody. Immunoprecipitated TRF2 proteins were recovered from the gel, and analysed by LC-MS/MS. In gel, digestion was performed as described previously . The peak at 126.09 m /z (immonium ion) is due to the presence of the acetylated lysine residue. ( E ) HEK293 cells were co-transfected with p300-HA and various point mutant constructs of Flag-TRF2 and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblotting with anti-acetylated lysine antibody. ( F ) HEK293 cells were co-transfected with p300-HA and either Flag-TRF2 or Flag-K293R and subjected to immunoprecipitation with anti-HA or anti-Flag antibodies, followed by immunoblotting with anti-Flag or anti-HA antibodies.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Transfection, Immunoprecipitation, Western Blot, In Vitro, Acetylation Assay, Purification, Staining, Autoradiography, Tandem Mass Spectroscopy, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Construct

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: p300 regulates the levels of endogenous TRF2. ( A ) HEK293 cells expressing p300-HA or p300ΔHAT-HA were subjected to immunoblotting as indicated. ( B ) HEK293 cells expressing p300 siRNA (sip300) or scrambled control siRNA (siControl) were subjected to immunoblotting as indicated. ( C ) HEK293 cells expressing p300-HA or p300 siRNA were treated with 100 μg/ml cycloheximide and together with, or without, 10 μM MG132 for the indicated times, followed by immunoblotting with anti-TRF2 or anti-actin antibodies. ( D ) Graphical representation of the relative TRF2 levels normalized against the β-actin loading control. The TRF2 expression levels were quantified with the average and standard deviation from three independent experiments. ( E ) HEK293 cells were co-transfected with Flag-TRF2 or Flag-K293R, TIN2-V5 and p300-HA, and subjected to immunoprecipitation with anti-V5 or anti-Flag antibodies, followed by immunoblotting with anti-Flag or anti-V5 antibodies. The p300 expression was detected with anti-p300 or anti-HA antibodies.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Expressing, Western Blot, Standard Deviation, Transfection, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: Acetylation inhibits ubiquitin-dependent proteolysis of TRF2. ( A ) HEK293 cells expressing Flag-TRF2 or Flag-K293R were treated with 100 μg/ml cycloheximide and together with, or without, 10 μM MG132 for the indicated times, followed by immunoblotting with anti-Flag or anti-actin antibodies. ( B ) Graphical representation of the relative TRF2 levels normalized against the β-actin loading control. The TRF2 expression levels were quantified with the average and standard deviation from three independent experiments. ( C ) HEK293 cells were co-transfected with HA-ubiquitin and either Flag-TRF2 or Flag-K293R and treated with, or without, 10 μM MG132 for 2 h as specified. Immunoprecipitation was carried out with anti-Flag antibody before probing with anti-HA antibody. ( D ) ChIP of telomeric DNA by ectopically expressed TRF2. HEK293 cells were co-transfected with Flag-TRF2 or Flag-K293R and together with p300-HA or p300 siRNA and subjected to ChIP with anti-Flag antibody. Total DNA and immunoprecipitated DNA were applied to nitrocellulose in a slot blot manifold. Duplicate slot blots were hybridized with a telomeric probe or an Alu probe. Input DNA represents 10% of total DNA. HEK293 cells were co-transfected with Flag-TRF2 or Flag-K293R and together with p300-HA or p300 siRNA and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblotting with anti-acetylated lysine antibody. The p300 expression was detected with anti-p300 or anti-HA antibodies. ( E ) Quantification of blots shown in D. Histogram values represent the expressed TRF2 telomeric ChIP signal normalized to input signal. The percentage of telomeric DNA in ChIP is shown with the average and standard deviation from three independent experiments. ( F ) ChIP of telomeric DNA by endogenous TRF2. HEK293 cells were transfected with p300-HA or p300 siRNA and subjected to ChIP with anti-TRF2 antibody. HEK293 cells expressing p300-HA or p300 siRNA were subjected to immunoprecipitation with anti-TRF2 antibody, followed by immunoblotting with anti-acetylated lysine antibody. The endogenous TRF2 expression was detected with anti-TRF2 antibody. ( G ) Quantification of blots shown in F. Histogram values represent the endogenous TRF2 telomeric ChIP signal normalized to input signal. The percentage of telomeric DNA in ChIP is shown with the average and standard deviation from three independent experiments.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Expressing, Western Blot, Standard Deviation, Transfection, Immunoprecipitation, Dot Blot

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: Overexpression of K293R induces a DNA-damage signal at telomeres. ( A ) HT1080 cells were transfected with TRF2-Myc or K293R-Myc, and multiple independent stable clones were isolated, followed by immunoblotting with anti-TRF2 or anti-actin antibodies. ( B ) HT1080 cells stably expressing TRF2-Myc or K293R-Myc were analysed by indirect immunofluorescence for co-localization of 53BP1 foci (green) with telomeric sites marked by TTAGGG-specific FISH probe (red). Representative fluorescence images of nuclei showing a large number of 53BP1 foci are shown as indicated. DNA was stained with DAPI (blue). A subset of 53BP1 foci co-localized with TTAGGG probe is indicated by arrows. ( C ) Quantification of the induction of 53BP1 foci by K293R overexpression. The average percentage of cells with either 6–10 or >10 53BP1 foci is shown. For each condition, at least 200 cells were counted. ( D ) Quantification of the induction of TIFs by K293R overexpression. The average percentage of cells with either 4–6 or >6 TIFs is shown. Cells with four or more DNA-damage foci co-localized with TTAGGG probe were scored as TIF positive. ( E ) The average percentage of 53BP1 foci located at telomeres was determined in TIF-positive nuclei.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Over Expression, Transfection, Clone Assay, Isolation, Western Blot, Stable Transfection, Expressing, Immunofluorescence, Fluorescence, Staining

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: Overexpression of K293R increases the occurrence of telomeric signal-free chromatid ends. ( A ) Representative telomere FISH analysis on metaphase spreads for telomere defects. HT1080 cells stably expressing the empty vector TRF2-Myc or K293R-Myc were processed for telomeric FISH. Telomere signal-free chromatid ends are indicated by arrows. ( B ) Quantification of telomere signal-free chromatid ends in HT1080 cells stably expressing the empty vector, TRF2-Myc or K293R-Myc.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Over Expression, Stable Transfection, Expressing, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: Overexpression of K293R limits cell proliferation. ( A ) Cell growth curves of HT1080 cell lines stably expressing the empty vector, TRF2-Myc or K293R-Myc. Stable cells were replated every 3 days to maintain log-phase growth and calculate the growth rate, with day 0 representing the first day after blasticidin selection. ( B ) Cell growth curves of DU145 cell lines stably expressing the empty vector, TRF2-Myc or K293R-Myc. ( C ) HT1080 cell lines stably expressing the empty vector, TRF2-Myc or K293R-Myc, at the indicated PDs were photographed after staining for SA-β-Gal activity. ( D ) The percentage of total cells that are positive for SA-β-Gal activity is shown in four HT1080 cell lines. For quantitation of senescent cells, a total of 200 cells were counted in each field and four fields were examined. ( E ) Flow cytometric analysis of HT1080 cell lines stably expressing the empty vector, TRF2-Myc or K293R-Myc. Cell lines at the indicated PDs were stained with propidium iodide, followed by fluorescence-activated cell sorter analysis. The percentage of total cells in each phase of the cell cycle is shown. Results are representative of three separate experiments. ( F ) The percentage of total cells in the aneuploidy fraction is shown with the average and standard deviation from three experiments being present in E.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Over Expression, Stable Transfection, Expressing, Plasmid Preparation, Selection, Staining, Activity Assay, Quantitation Assay, Fluorescence, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: Regulation of Telomeric Repeat Binding Factor 1 Binding to Telomeres by Casein Kinase 2-mediated Phosphorylation

doi: 10.1074/jbc.m710065200

Figure Lengend Snippet: FIGURE 1. Physical interaction between TRF1 and CK2. A, analysis of the physical interaction between TRF1 and CK2 using the yeast two-hybrid assay. RAP1 and unrelated FKBP52 were used as TRF2-binding and negative controls, respectively. The blue signal on the SG-HWU/X plate and the growth on the SG-HWUL plate indicate activation of the reporter genes, LacZ and LEU2, respectively. S, synthetic; G, galactose; H, histidine (); W, tryptophan (); U, uracil, (); L, leucine (); X, X-Gal (5-bromo-4-chloro-3-indolyl--D- galactopyranoside). B, interaction between TRF1 and CK2 in vitro. GST-TRF1, GST-TRF2, or GST were immobilized on glutathione-Sepharose and incu- bated with MCF7 cell extracts, followed by detecting endogenous CK2 by immunoblotting. C, coimmunoprecipitation of CK2 and TRF1. MCF7 cells were transfected with FLAG-TRF1 or FLAG-TRF2 and then subjected to immu- noprecipitation as indicated, followed by immunoblotting with anti-CK2 antibody. Right, MCF7 cells transfected with FLAG-TRF1 or FLAG-TRF2 were subjected to immunoprecipitation with anti-CK2 antibody, followed by immunoblotting with anti-FLAG antibody. D, MCF7 cells were subjected to immunoprecipitation with either anti-TRF1 or anti-TRF2 antibodies, followed by immunoblotting with anti-CK2 antibody. Endogenous levels of TRF1 and TRF2 in MCF7 cells were detected. E, GST-TRF1 was incubated with purified CK2 and [-32P]ATP, and the radiolabeled proteins were separated on an 8% SDS-polyacrylamide gel and autoradiographed. As controls, CK2 was incu- batedwithGSTorGST-TRF2asindicated.ThepositionsofGST-TRF1andCK2 and - subunits are indicated. Right, GST fusions were visualized by Coomas- sie Blue staining. Molecular mass makers (in kilodaltons) are indicated. F, GST- TRF1 was incubated with purified CK2 and [-32P]ATP in the presence of increasing concentrations of DRB as indicated. The radiolabeled proteins were separated and autoradiographed.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF1 (Sigma), anti-TRF2 (Upstate), antiCK2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-FLAG (Sigma), anti-V5 (Invitrogen), anti-HA (SantaCruzBiotechnology), and anti-RFP (Clontech) antibodies.

Techniques: Y2H Assay, Binding Assay, Activation Assay, In Vitro, Western Blot, Transfection, Immunoprecipitation, Incubation, Purification, Staining

Journal: Journal of Biological Chemistry

Article Title: Regulation of Telomeric Repeat Binding Factor 1 Binding to Telomeres by Casein Kinase 2-mediated Phosphorylation

doi: 10.1074/jbc.m710065200

Figure Lengend Snippet: FIGURE 3. Inhibition of CK2 by DRB leads to ubiquitination and degradation of TRF1. A, MCF7 cells trans- fected with FLAG-TRF1 or FLAG-TRF2 were treated with 200 M DRB for the indicated times. Lysates were resolved on an 8% SDS-PAGE and analyzed by immunoblotting using anti-FLAG antibody. Cells were pre- treatedwith10MMG132for2h,eitheraloneorbeforetreatmentwithDRB.B,MCF7cellswereincubatedwith 100 g/ml cycloheximide and together with or without 200 M DRB for the indicated times. Cells were incu- bated for 3 h with 100 g/ml cycloheximide and 10 M MG132. C, graphical representation of relative TRF1 levels normalized against the -actin loading control. The graph represents an average of three experiments. D, MCF7 cells expressing HA-ubiquitin were untreated or treated for 4 h with 200 M DRB or 10 M MG132, or a combination of the two as specified. Immunoprecipitation was performed with anti-TRF1 or anti-TRF2 anti- bodies before probing with anti-HA antibody. E and F, MCF7 cells were untreated or treated with 200 M DRB for 4 h and analyzed by indirect immunofluorescence. Cells were pretreated with 10 M MG132 for 2 h before treatment with DRB. Paraformaldehyde-fixed cells were stained with anti-TRF1 or anti-TRF2 and anti-CK2 antibodies. 4,6-Diamino-2-phenylindole (DAPI) staining of DNA is shown in blue.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF1 (Sigma), anti-TRF2 (Upstate), antiCK2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-FLAG (Sigma), anti-V5 (Invitrogen), anti-HA (SantaCruzBiotechnology), and anti-RFP (Clontech) antibodies.

Techniques: Inhibition, Ubiquitin Proteomics, SDS Page, Western Blot, Control, Expressing, Immunoprecipitation, Immunofluorescence, Staining

Journal: Journal of Biological Chemistry

Article Title: Regulation of Telomeric Repeat Binding Factor 1 Binding to Telomeres by Casein Kinase 2-mediated Phosphorylation

doi: 10.1074/jbc.m710065200

Figure Lengend Snippet: FIGURE 4. CK2 siRNA depletion caused a reduction in the level of endog- enous TRF1. A, immunoblots showing the levels of CK2 or CK2 before and after knockdown by siRNA. MCF7 cells were transfected for 3 days with siRNA and visualized with anti-CK2 or anti-CK2 antibodies. The CK2 levels were determined from four independent experiments using -actin as a loading control. B, endogenous TRF proteins in MCF7 cells transfected with siRNAs specific for CK2 or CK2 were detected using anti-TRF1 and anti-TRF2 anti- bodies. C, indirect immunofluorescence analysis of MCF7 cells transfected with CK2 siRNA or control siRNA, visualized with anti-TRF1 (red) and anti- CK2 (green) antibodies. D, indirect immunofluorescence analysis of cells transfected with CK2 siRNA and control siRNA.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF1 (Sigma), anti-TRF2 (Upstate), antiCK2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-FLAG (Sigma), anti-V5 (Invitrogen), anti-HA (SantaCruzBiotechnology), and anti-RFP (Clontech) antibodies.

Techniques: Western Blot, Knockdown, Transfection, Control, Immunofluorescence

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Schematic representation of luciferase screening approach. Upper panel shows the four target predictions software used for in silico analysis. Bottom panel indicates the main steps performed in the high‐throughput screening. B Upper panel, sequence interaction of miR‐182‐3p with the target site of the wild type 3′UTR of TRF2 in human. Bottom panel, generation of mutant 3′UTR of TRF2 luciferase construct containing the deletion of target site for miR‐182‐3p. C–E Luciferase reporter assay in HeLa cells using the synthetic miR‐Control or miR‐182‐3p in combination with the wild type (C) or the mutant 3′UTR of TRF2 construct (D) or the wild type 3′UTR of TRF1 (E). F, G Western blotting for TRF2 expression in telomerase‐positive (HeLa, HCT116, MDA‐MB‐231, MDA‐MB‐436) and ALT‐positive (U2‐OS, Saos‐2) cells transiently transfected with miR‐Control or miR‐182‐3p. Upper panel shows the quantification of TRF2 expression. Bottom panel, representative images are shown, actin was used as loading control. H U2‐OS cells transiently transfected with the miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor were assayed by quantitative immunofluorescence for TRF2 3 days post‐transfection. Left panel, representative images. Scale bar: 10 μm. Right panel, quantification of TRF2 fluorescence intensity. a.f.u. arbitrary fluorescence units. N = number of analyzed nuclei. Red bar indicates mean value. I U2‐OS cells transfected as described in (H) were assayed by immunofluorescence combined with telomeric FISH. Left panel, representative images of co‐localizations between TRF2 and telomeres (white arrowheads). Scale bar: 10 μm. Right panel, co‐localizations were analyzed using ImageJ software. N = number of analyzed nuclei. Data information: For (C–G and I), data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by Student's t ‐test; for (H), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Luciferase, Software, In Silico, High Throughput Screening Assay, Sequencing, Mutagenesis, Construct, Reporter Assay, Western Blot, Expressing, Transfection, Immunofluorescence, Fluorescence, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Results of high‐throughput luciferase screening performed in Hela cells using the wild type 3′UTR‐TRF2 vector in combination with each of the 54 miRNAs selected by in silico analysis. Three days post‐transfection, luciferase ratio (Renilla:Firefly) of each miRNA was calculated, the control miRNA was set “1.” Renilla:Firefly ratios < 1 indicate target specificity of candidate miRNAs for the 3′UTR of TRF2. miRNAs near to the ratio of 0.5 were considered for further analysis. Two biological replicates were performed. B HeLa cells transiently transfected with the indicated miRNAs (miR‐Control, miR‐182‐3p, miR‐519e‐5p, miR‐296‐3p) were assayed by western blotting. Upper panel, quantification of TRF2 expression. Bottom panel, representative images of TRF2, TRF1 and RAP1 are shown, actin was used as loading control. C Analysis of TRF2 mRNA expression performed by qPCR in four different cancer cell lines (HeLa, MDA‐MB‐231, MDA‐MB‐436, U2‐OS) 3 days post‐transfection with miR‐Control or miR‐182‐3p. The control miRNA was set “1.” Three independent experiments were performed. D, E Telomeric ChIP assay in MDA‐MB‐231 (D) and U2‐OS cells (E). Quantification of TRF2 enrichment at telomeric repeats, in the different conditions, is shown in the table under the respective figure. Alu probe and Rabbit IgG were used as negative control for the assay. Data information: For (A), data are presented as mean values. For (B, C), data are presented as mean values ± SD and Student t‐ test was used to calculate statistical significance. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: High Throughput Screening Assay, Luciferase, Plasmid Preparation, In Silico, Transfection, Western Blot, Expressing, Negative Control

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A MDA‐MB‐231 cells were transiently transfected with the indicated miRNAs or siRNA. The indicated DNA damage markers were assayed by western blotting. Actin was used as loading control. B Telomeric DNA FISH performed in MDA‐MB‐231 transiently transfected with the indicated miRNAs. Telomere length was measured by TLF software and indicated as arbitrary fluorescence unit (a.f.u). N = number of analyzed nuclei. Black bar indicates mean value. C DNA damage markers were assayed by western blotting in HeLa cells. Actin was used as loading control. D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. Co‐localizations of γH2AX with telomeres are indicated as mean number of TIFs per nucleus. E Representative images and enlargements of co‐localizations of experiment described in D. F Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. G Representative images of co‐localizations relative to the experiment described in (F). H, I MDA‐MB‐231 and HeLa cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with miR‐Control or miR‐182‐3p. TRF2, pATM and γH2AX expression were assayed by western blotting. Actin was used as loading control. Data information: For (D) and (F), data are presented as mean values ± SD. Three independent replicates were performed. Scale bar: 10 μm. At least 60 nuclei were analyzed in (D) and (F). A Student t‐ test was used to calculate statistical significance. For (B), P values are determined by Mann–Whitney t ‐test. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Western Blot, Software, Fluorescence, Immunofluorescence, Expressing, Plasmid Preparation, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: Immunofluorescence analysis of γH2AX combined with telomeric FISH (TIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The mean number of TIFs per nucleus was analyzed. Representative images and enlargements of co‐localizations (white arrowheads) relative to the experiment described in (A). Scale bar: 10 μm. Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images of co‐localizations (white arrowheads) relative to the experiment described in (C). Scale bar: 10 μm. Quantification of TIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The mean number of TIFs per nucleus was quantified. Representative images and enlargements relative to the experiment described in (E). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Quantification of PIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images relative to the experiment described in (G). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Data information: For (A, C, E, G) data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. At least 60 nuclei were analyzed for each experimental condition. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐436 and MDA‐MB‐231 cells underwent two rounds of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Starting from the day of the second transfection, cell confluence was monitored by Incucyte every 24 h up to a maximum of 3 days. The percentage of cell confluence was analyzed. C, D Cell number of MDA‐MB‐436 (C) and MDA‐MB‐231 (D) cells and TRF2 expression were analyzed by automatic cell count and by western blotting at the end of the experiment described in (A) and (B). Actin was used as loading control. E Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐436 at the end of the second cycle of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Red boxes indicate early and late apoptotic cells. F Quantification of Annexin V‐positive cells (%) of experiment described in (E). G Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐231 as described in (E). H Quantification of Annexin V‐positive cells (%) of experiment described in (G). I, J MDA‐MB‐436 cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with indicated miRNAs and cell count (I) or apoptosis (J) analysis was performed 72 h post‐transfection. Data information: For (A, B) data are shown as mean ± SEM. For (C, D, F, H, I, J), data are shown as mean ± SD. For (A–D) and (I), three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. For (F), (H) and (J), two different biological replicates were performed. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Cell Counting, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Western blotting for TRF2 expression in BJ cells transiently transfected with miR‐Control or miR‐182‐3p. The graph represents the quantification of three independent experiments. Representative images are shown, Actin was used as loading control. Unspecific bands are indicated with (*). B, C Mean of γH2AX foci per nucleus was analyzed in BJ cells 72 h post‐transfection with the indicated miRNAs. Representative images of γH2AX foci are shown in (C). D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in BJ cells 72 h post‐transfection with the indicated miRNAs. Left panel: The mean number of TIFs per nucleus was analyzed. Right panel: Representative images and enlargements of co‐localizations. E Cell number of BJ cells was analyzed by automatic cell count at the end of the second round of transfection with miR‐Control or miR‐182‐3p. F FACS analysis to evaluate cell cycle progression by Propidium Iodide (PI) staining in BJ cells treated as indicated in (E). G β‐Galactosidase assay in BJ cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. H–J IL‐6 (H), CXCL1 (I), IL‐8 (J) factors were analyzed by ELISA to evaluate the senescence‐associated secretory phenotype (SASP) in BJ cells treated as indicated in (G). Data information: For (A, B, D, E and G–J), a student t‐ test was used to calculate statistical significance. Scale bars (10 μm). P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Western Blot, Expressing, Transfection, Immunofluorescence, Cell Counting, Staining, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A TRF2 and γH2AX expression after two rounds of transfection with the indicated miRNAs, was analyzed by western blotting in MCF10A cells. Actin was used as loading control. B–E The mean number of γH2AX foci (B) and TIFs (D) per nucleus were analyzed 72 h post‐transfection with the indicated mimic miRNAs in MCF10A cells. Representative images (C) and (E) are referred to the experiment showed in (B) and (D) respectively. F, G Cell confluence (F) of MCF10A was monitored by Incucyte, every 24 h starting from the day of the second transfection, and cell number (G) was counted at the end of experiment (day 4). H–I Cell cycle progression analysis by PI staining (H) and cell death analysis by Annexin V assay (I) were performed in MCF10A upon two rounds of transfection with the indicated miRNAs. J β‐Galactosidase assay in MCF10A cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. Data information: Panels (B, D, F, G, J) data are presented as mean values ± SD. A Student t‐ test was used to calculate statistical significance. P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Transfection, Western Blot, Staining, Annexin V Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐231 (A) and MDA‐MB‐436 (B) tumor xenografts were treated with LNPs‐empty, LNPs‐miR‐Control or by LNPs‐miR‐182‐3p when the tumors became palpable. Mice were treated 6 times by intravenous tail vein injections with 20 μg of LNPs‐miR‐Control, LNPs‐miR‐182‐3p or equivalent volume of LNPs‐empty as indicated in the scheduling. The mean of tumor volumes ( n = 5 per group) is shown. C, D Tumors from mice treated in (A) and (B) were processed to measure miR‐182‐3p expression by TaqMan qPCR. E Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA‐MB‐231 human breast cancer xenografts. Scale bar: 50 μm. F The histograms show the expression of TRF2, calculated as immunoreactivity score (IRS) by IHC, and the count of positive cells to γH2AX, TUNEL or CD31 staining. The analyses were performed on three mice per group, and the points represent the number of field analyzed for each condition. G, H Luminescent MDA‐MB‐436 cells were injected into the brain and monitored by IVIS imaging system. After 1 week from implant, treatment with LNPs‐miR‐Control and LNPs‐miR‐182‐3p was performed as indicated in (A) and (B). Representative images from in vivo (upper panel) or ex‐vivo (bottom panel) brain tumors are shown in (G). Boxplots (H) show the measurement of photons for each brain tumor ( n = 5 per group) acquired at the indicated times. Data information: For (A, B, F), data are shown as mean ± SD. For (C, D, H), the line in the middle of the box plot denotes a median value, the limits of box represent the interquartile range (25 th to 75 th percentiles), while, the whiskers denote the minimum to maximum values. For (A–D) and (H), P values are determined by unpaired two‐tailed t‐ test; for (F), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, TUNEL Assay, Staining, Injection, Imaging, In Vivo, Ex Vivo, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: The organs (brain, liver, kidney) taken from mice, previously engrafted with MDA‐MB‐231 cells and treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p, were assayed for miR‐182‐3p expression by TaqMan qPCR. Representative images show IHC analysis on tumor samples, from mice bearing MDA‐MB‐436 human breast cancer xenografts, with the indicated markers. Scale bar: 50 μm. The histograms show the expression of TRF2 indicated as immunoreactivity score (IRS) and the percentage of positive cells to γH2AX, TIUNEL or CD31 staining in MDA‐MB‐436 xenografts. Three mice per group were analyzed, the points represent the number of field analyzed for each condition. Data information: For (A, C), data are presented as mean values ± SD. Statistical significance using unpaired (A) or Mann–Whitney t‐ test (C) was calculated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B PDTCs #1 and #2 underwent two rounds of transfection with miR‐Control or miR‐182‐3p. Three days after the second transfection, miR‐182‐3p and TRF2 expression were analyzed by TaqMan qPCR and western blotting, respectively. Actin was used as loading control. C, D Left panel, area of each PDTCs was measured by ImageJ. Right panel, representative images are shown. Scale bar: 50 μm. At least 85 3D cells were analyzed for each experimental condition. E NSG mice implanted with breast PDTX (#2) were treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p as indicated in the scheduling. Caliper measurement of tumors was taken at the indicated days. The mean of tumor volumes ( n = 5 per group) is shown. F miR‐182‐3p expression of tumors from mice treated in (E) was assayed by TaqMan qPCR. G Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in (E). Scale bar: 50 μm. H The histograms show the expression levels of TRF2 measured as immunoreactivity score (IRS), the percentage of positive cells to γH2AX and TUNEL. The analysis was performed on three mice per group, the points represent the number of field analyzed for each condition. Data information: For (A–F) and (H), data are shown as mean ± SD. For (A–F), P values are determined by unpaired two‐tailed t‐ test; for (H), P values are determined by Mann–Whitney t ‐test. For the experiments showed in (A, B) and (C, D) two or three biological replicates were performed, respectively. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Western Blot, TUNEL Assay, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Representative images of intestine sections from mice previously treated with LNPs‐Empty or LNPs‐miR‐182‐3p. H&E staining (scale bar: 200 μm) and IHC analysis with TRF2 or γH2AX antibodies are shown (scale bar: 50 μm). B, C Quantification of TRF2 expression as immunoreactivity score (IRS) (B) and of γH2AX‐positive cells (%) (C) on intestine samples. D Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of skin samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). E, F Quantification of TRF2 expression as immunoreactivity score (IRS) (E) and of γH2AX‐positive cells (%) (F) on skin samples. G Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of bone marrow samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). H, I Quantification of TRF2 expression as immunoreactivity score (IRS) (H) and of γH2AX‐positive cells (%) (I) on bone marrow samples. Data information: For (B, C, E, F, H, I), data are shown as mean ± SD. A Mann–Whitney test t‐ test was used to calculate statistical significance. Four mice per group were analyzed, the points represent the number of field analyzed for each condition. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: Immunity & Ageing : I & A

Article Title: Topological DNA damage, telomere attrition and T cell senescence during chronic viral infections

doi: 10.1186/s12979-019-0153-z

Figure Lengend Snippet: PCR primers used in this study

Article Snippet: Primary and secondary antibodies included Top1, PARP1, γH2AX, TRF1, TRF2, TPP1, TIN2, RAP1, POT1, ATM, pATM, CHK2, ATR, GAPDH, β-actin, and horseradish peroxide-conjugated antibodies (Cell Signaling).

Techniques: