Journal: Oncoimmunology

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

doi: 10.1080/2162402X.2021.1901446

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

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

Techniques: Staining

Journal: Oncoimmunology

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

doi: 10.1080/2162402X.2021.1901446

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

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

Techniques: Biomarker Discovery, Mutagenesis

Journal: Oncoimmunology

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

doi: 10.1080/2162402X.2021.1901446

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

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

Techniques: Biomarker Discovery

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-17A Orchestrates Lung Injury and Remodeling Through p53 and uPA System Crosstalk

doi: 10.3390/ijms27041841

Figure Lengend Snippet: Regulation of BLM-LI and TSE-LI by IL-17A. ( A ) Lung sections of WT mice exposed to saline or BLM for 24–72 h were subjected to IHC staining for IL-17A antigen ( i ). AECs isolated from WT and IL-17A-deficient mice exposed to saline or BLM were analyzed for apoptosis and Sirt1 expression by Western blotting (WB) three days later ( ii ). Lung sections of WT and IL-17A −/− mice exposed to saline or BLM were subjected to Masson’s trichrome staining 21 days later ( iii ). Whole lung homogenates of WT and IL-17A −/− mice exposed to saline or BLM as in were analyzed for total HYP content ( iv ) or for Col-1 and FN by WB 21 days after BLM ( v ). ( B ) WT, p53 −/− and PAI-1 −/− mice were kept in ambient air or exposed to passive TS for 20 weeks. Total lung homogenates of control mice kept in ambient air, TSE WT mice, and p53 −/− or PAI-1 −/− mice were analyzed for IL-17A and IL-17RA protein by WB ( i ). Total RNA from mice kept in ambient air, TSE WT mice, and p53 −/− or PAI-1 −/− mice was analyzed for IL-17A mRNA ( ii ). AECs from TSE WT and IL-17A −/− mice were analyzed for p53, ACp53, cleaved (Cl.) caspase-3/caspase-3 and Sirt1 by WB ( iii ). AECs from lungs of IL-17A −/− mice kept in ambient air or TSE, as well as mice treated with or without CSP7 or CP, were analyzed for telomere length by qPCR ( iv ). AECs from lungs of IL-17A −/− mice treated as in ( B(iv )) were immunoblotted for the listed proteins ( v ). Lung sections of IL-17A −/− mice treated as in ( B ( iv )) were subjected to IHC for telomere binding shelterin complex (TRF1 and TRF2) proteins ( vi ).

Article Snippet: 12 , TRF2 (D1Y5D) rabbit mAb , CST , 13136S , 1:1000 , 1:500.

Techniques: Saline, Immunohistochemistry, Isolation, Expressing, Western Blot, Staining, Control, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: c-Myc Quadruplex-forming Sequence Pu-27 Induces Extensive Damage in Both Telomeric and Nontelomeric Regions of DNA *

doi: 10.1074/jbc.M113.505073

Figure Lengend Snippet: Pu-27 induces phosphorylation of H2AX (γH2AX). U937 cells and ΔB-U937 cells treated with Pu-27 and were subjected to Western blot and FACS analysis. A, 3-day treatment of Pu-27 showed profound up-regulation of γH2AX expression in U937 cells but almost no changes in dB-U937 cells. Relative expressions of TRF2 in U937 and ΔB-U937 cells were also shown. TRF2 expression is down-regulated in U937 cells and no changes in ΔB-U937 cells when treated with Pu-27 (consistent with RT-PCR data in Fig. 6). Bottom panel, relative expression presented in bar graphs. *, p < 0.05. B, by FACS analysis γH2AX has shown continued to be highly expressed from days 1–3, and again there was no significant change in ΔB-U937 cells. Bottom panels, relative expression presented in bar graphs. *, p < 0.05.

Article Snippet: Aliquots of 0.4 ml of cells were transfected with 2 μg of the plasmid pLPC TRF2 ΔB (Addgene), which contains the TRF2 gene in which the basic domain of amino acids 1–44 is deleted.

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: c-Myc Quadruplex-forming Sequence Pu-27 Induces Extensive Damage in Both Telomeric and Nontelomeric Regions of DNA *

doi: 10.1074/jbc.M113.505073

Figure Lengend Snippet: Pu-27 inhibits the molecules related to DNA damage repair machinery in U937. A, RT-PCR of U937 and ΔB-U937 cells treated with Pu-27 for 3 days. Pu-27 inhibits ATM, RAD17, RAD50, CHK14, and CHK2 but not H2AX, BRCA1, and telomerase reverse transcriptase in U937 cells. B, ΔB-U937 cells showed no changes of TRF2, TRF1, POT1, TIN2, RAD17, RAD50, and 53BP1; down-regulation of ATM; up-regulation of CHK1 and CHK2; and H2AX and BRCA1.

Article Snippet: Aliquots of 0.4 ml of cells were transfected with 2 μg of the plasmid pLPC TRF2 ΔB (Addgene), which contains the TRF2 gene in which the basic domain of amino acids 1–44 is deleted.

Techniques: Reverse Transcription Polymerase Chain Reaction

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: Molecular Biology of the Cell

Article Title: Telomeric DNA Mediates De Novo PML Body Formation

doi: 10.1091/mbc.E09-04-0309

Figure Lengend Snippet: Endogenous PML, Sp100 and Hausp accumulate at telomeric sites but not at centromeres in U2OS cells recovering from MMS treatment. (A) Immunofluorescence image of a U2OS cell treated with MMS, fixed and stained with anti-PML (green) and anti-TRF2 (red) antibodies. (B) Image of a U2OS cell that recovers from MMS treatment and is stained with antibodies against PML (green) and TRF2 (red). (C) Image of a HeLa cell that recovers from MMS treatment and is stained with antibodies against PML (green) and TRF2 (red). Arrows in B and C indicate the positions where PML colocalize or associate with TRF2 foci. (D) Localization of Sp100 at telomeric sites in a U2OS cell that recovers from MMS treatment. During recovery from MMS treatment, U2OS cells were fixed and stained with anti-Sp100 and anti-TRF2 antibodies. (E) Immunofluorescence image of a U2OS cell that recovers from MMS treatment. Sites where Hausp colocalize with telomeric DNA are indicated by arrows. (F) PML does not colocalize with centromeres in a U2OS cell that recovers from MMS treatment. After MMS treatment, U2OS cells were incubated in fresh medium, fixed and stained with anti-PML (green) and anti-CENPA (red) antibodies. All cell nuclei are counterstained with DAPI (blue).

Article Snippet: The following antibodies were used for immunofluorescence staining: mouse mAb 5E10 against PML (gift from R. van Driel, Amsterdam, The Netherlands), rabbit polyclonal antibody against PML (1130 directed against sequence: MEPAPARSPRPQQDP), rabbit polyclonal antibody against SP100 (ab1380, Chemicon, Temecula, CA), rabbit polyclonal antibody against Daxx (sc-7152, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal antibody against Hausp (A300–033A, Bethyl Laboratories, Montgomery, TX), mouse mAb against TRF1 (ab10579–50, Abcam, Cambridge, MA), mouse mAb against TRF2 (IMG-124, Imgenex, San Diego, CA), human autoimmune serum against centromeres (Antibodies Incorporated, Davis, CA), rabbit polyclonal antibody against γH2AX (A300–081A, Bethyl Laboratories), rabbit polyclonal antibody against 53BP1 (NB100–304, Novus Biologicals, Littleton, CO), and rabbit polyclonal antibody against SMC5 (A300–236A, Bethyl Laboratories).

Techniques: Immunofluorescence, Staining, Incubation