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: Biochimica et biophysica acta. Molecular cell research

Article Title: TRF2 recruits ORC through TRFH domain dimerization.

doi: 10.1016/j.bbamcr.2016.11.004

Figure Lengend Snippet: Fig. 1. Co-localization of ORC1 with TRF2-LacI foci in U2OS 2-6-3 cells. (A) U2OS or U2OS 2-6-3 cells were transfected with the empty or HA-LacI expression vector for 24 h and then immunostained with anti-LacI antibody or control mouse IgG (red), followed by DAPI staining (blue). Representative images are shown. Scale bars, 20 μm. (B) U2OS 2-6-3 cells transfected with the TRF2-LacI expression vector were immunostained with anti-LacI antibody (red) and counterstained with DAPI (blue). (C and D) U2OS 2-6-3 cells were co- transfected with the expression vectors indicated (left) and immunostained for HA, TRF2, or ORC1 (red). GFP-LacI foci are highlighted with dotted frames. Yellow frames indicate co-lo- calization of HA-LacI, TRF2, or ORC1 to the foci of GFP-LacI and white frames indicate non-co-localization. Co-localization frequency (%) of the HA-LacI, TRF2, or ORC1 foci with the GFP foci is depicted. ***, p b 0.001 (χ2-test).

Article Snippet: The other antibodies were purchased from different suppliers: Rabbit Normal IgG (DAKO, Product No. X0903),MouseNormal IgG (Southern Biotech, 0107-01), LacI (clone 9A5, Merck Millipore), HA-tag (clone 3F10, Roche), Flag-tag (clone M2, Sigma), TRF2 (clone 4A794, Merck Millipore), ORC3 (clone 1D6, Santa Cruz), HP1α (Cell Signaling, #2616), H3K9me3 (Merck Millipore, 07- 442), Alexa594 Donkey anti-Rabbit IgG (Thermo Fisher), CF594 Donkey anti-Rat IgG (Biotium), CF594 Goat anti-Mouse IgG (Biotium), and CF488 Goat anti-Rabbit IgG (Biotium).

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Staining

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: TRF2 recruits ORC through TRFH domain dimerization.

doi: 10.1016/j.bbamcr.2016.11.004

Figure Lengend Snippet: Fig. 3. The TRFH domain of TRF2 is sufficient for interaction with ORC. (A) Schematic representation of the full-length TRF2 (FL) and the TRF2 N- and C-terminal truncated mutants. Summary of their ability to recruit ORC1 onto the lacO array is shown on the right (for details, see Table 1). (B) HEK293T cells were co-transfected with Flag-ORC1 and LacI-fusion proteins, as indicated, for 42 h. After cross-linking with formaldehyde and solubilization, cell lysates were immunoprecipitated with anti-LacI antibody. Immunoprecipitates (IPs) and 1% of the input were immunoblotted with the indicated antibodies. Asterisks indicate the position of each LacI-fused protein. The data are representative of three independent experiments, which showed similar results.

Article Snippet: The other antibodies were purchased from different suppliers: Rabbit Normal IgG (DAKO, Product No. X0903),MouseNormal IgG (Southern Biotech, 0107-01), LacI (clone 9A5, Merck Millipore), HA-tag (clone 3F10, Roche), Flag-tag (clone M2, Sigma), TRF2 (clone 4A794, Merck Millipore), ORC3 (clone 1D6, Santa Cruz), HP1α (Cell Signaling, #2616), H3K9me3 (Merck Millipore, 07- 442), Alexa594 Donkey anti-Rabbit IgG (Thermo Fisher), CF594 Donkey anti-Rat IgG (Biotium), CF594 Goat anti-Mouse IgG (Biotium), and CF488 Goat anti-Rabbit IgG (Biotium).

Techniques: Transfection, Immunoprecipitation

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: TRF2 recruits ORC through TRFH domain dimerization.

doi: 10.1016/j.bbamcr.2016.11.004

Figure Lengend Snippet: Fig. 4. Dimerization-defective mutants of TRF2 exhibit a defect in the ORC recruitment. (A) Soluble extracts from the U2OS cells transfected with the indicated expression vectors were immunoprecipitated with anti-LacI antibody. The IPs and 3% of the input were immunoblotted with anti-LacI or anti-HA antibodies. (B) Bacterially produced recombinant TRF2 (45– 244) and TRF2 (45–244/DP) were subjected to gel filtration column chromatography. The indicated fractions were analyzed by SDS-PAGE, followed by silver staining. (C) U2OS 2-6-3 cells transfected with the indicated expression vectors were double-immunostained with anti-LacI antibody (red) and anti-ORC1 antibody (green), followed by DAPI staining and analysis as described in Fig. 2A. ***, p b 0.001 compared to HA-LacI in Fig. 2A (χ2-test). Scale bars, 20 μm. (D) GST or GST-TRF2 mutants were incubated with HeLa cell extracts. The bound proteins and 2.5% of the input were analyzed by Coomassie Brilliant Blue (CBB) staining or immunoblotting with the indicated antibodies. Asterisks indicate the position of GST or GST-fused pro- teins. (E) In vitro-translated ORC1 was incubated with GST or GST-TRF2 mutants. The pulled-down proteins and 25% of the input were analyzed by CBB staining or immunoblotting with anti-ORC1 antibody. Asterisks indicate the positions of GST or GST-fused proteins. (F) HEK293T cells were co-transfected with Flag-ORC1 and empty vector or HA-tagged TRF2 proteins, as indicated, for 42 h. After cross-linking with formaldehyde and solubilization, cell lysates were immunoprecipitated with anti-HA antibody. IPs and 1% of the input were immunoblotted with the indicated antibodies. The data (A–F) are representatives of at least two independent experiments, which showed similar results.

Article Snippet: The other antibodies were purchased from different suppliers: Rabbit Normal IgG (DAKO, Product No. X0903),MouseNormal IgG (Southern Biotech, 0107-01), LacI (clone 9A5, Merck Millipore), HA-tag (clone 3F10, Roche), Flag-tag (clone M2, Sigma), TRF2 (clone 4A794, Merck Millipore), ORC3 (clone 1D6, Santa Cruz), HP1α (Cell Signaling, #2616), H3K9me3 (Merck Millipore, 07- 442), Alexa594 Donkey anti-Rabbit IgG (Thermo Fisher), CF594 Donkey anti-Rat IgG (Biotium), CF594 Goat anti-Mouse IgG (Biotium), and CF488 Goat anti-Rabbit IgG (Biotium).

Techniques: Transfection, Expressing, Immunoprecipitation, Produced, Recombinant, Column Chromatography, SDS Page, Silver Staining, Staining, Incubation, Western Blot, In Vitro, Plasmid Preparation

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: TRF2 recruits ORC through TRFH domain dimerization.

doi: 10.1016/j.bbamcr.2016.11.004

Figure Lengend Snippet: Fig. 5. MCM7 loading is enhanced by dimerized TRF2 (45–244)-LacI. (A) U2OS 2-6-3 cells were transfected with the indicated expression vectors. Cells were first extracted with Triton X- 100 and then double-immunostained with anti-LacI antibody (red) and anti-MCM7 antibody (green), followed by DAPI staining and analysis as described in Fig. 2A. ***, p b 0.001 (χ2-test). Scale bars, 20 μm. (B) U2OS 2-6-3 cells were transfected with the expression vectors and then double-immunostained as indicated in the figure, followed by DAPI staining and analysis as described in Fig. 2A. *, p b 0.05 (χ2-test).

Article Snippet: The other antibodies were purchased from different suppliers: Rabbit Normal IgG (DAKO, Product No. X0903),MouseNormal IgG (Southern Biotech, 0107-01), LacI (clone 9A5, Merck Millipore), HA-tag (clone 3F10, Roche), Flag-tag (clone M2, Sigma), TRF2 (clone 4A794, Merck Millipore), ORC3 (clone 1D6, Santa Cruz), HP1α (Cell Signaling, #2616), H3K9me3 (Merck Millipore, 07- 442), Alexa594 Donkey anti-Rabbit IgG (Thermo Fisher), CF594 Donkey anti-Rat IgG (Biotium), CF594 Goat anti-Mouse IgG (Biotium), and CF488 Goat anti-Rabbit IgG (Biotium).

Techniques: Transfection, Expressing, Staining

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