Journal: Oncotarget

Article Title: Optogenetic regulation of site-specific subtelomeric DNA-methylation

doi: 10.18632/oncotarget.10394

Figure Lengend Snippet: a. Schematic of the HALO tagged TCON and ECON fusion proteins, b. Western blot analysis demonstrates production of intact TCON (~149 kD; lane-1), ECON (~223 kD; lane-2), and TCON plus ECON (lane-3) fusion proteins in HeLa cells. Representative images showing the nuclear localization of TCON c. ECON d. their co-localization e. and their overlap with DAPI stained nucleic acids f. g. Schematic showing the binding of TCON to the telomeric repeat sequences through TRF1. FCS measurements showed the distinct distribution and diffusion pattern of TCON tagged EGFP j, k. in comparison to the control EGFP h, i. Co-localization of TCON and TRF2 is revealed using the overlapping fluorescence of EGFP of TCON and Alexa-647 TRF2 (m) antibody bound to TRF2 l, m and n. in HeLa cells. Because TRF2 is telomere specific binding protein, the co-localization of TCON to the same locus reveals the association of TCON with telomeres.

Article Snippet: Blocking was performed with PBS solution containing 5% goat serum and 0.3% Triton X-100 for 1 h. Mouse monoclonal anti-TRF2 antibody (ab13579, Abcam) diluted 1:500 was incubated overnight with the cells at 4°C.

Techniques: Western Blot, Staining, Binding Assay, Diffusion-based Assay, Comparison, Control, Fluorescence

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: Nucleic Acids Research

Article Title: Aurora Kinase B, a novel regulator of TERF1 binding and telomeric integrity

doi: 10.1093/nar/gkx904

Figure Lengend Snippet: AURKB localization at telomere is linked to stem cell pluripotency. ( A ) AURKB localizes to the telomeres of mitotic mouse ES129.1 cells (arrowheads in (i)), but is lost in ES129.1 cells subjected to retinoic acid treatment differentiation (ii). Note that AURKB localization at pericentric heterochromatin is not lost in differentiated cells (arrows in Ai-ii). ( B ) AURKB localizes to the pericentric heterochromatin (arrows) but not to the telomeres of somatic, non-ESCs including mouse NIH3T3 (i) and human HT1080 (ii), telomerase-negative SKLU1 ALT cancer (iii) and telomerase overexpressing HT1080 (iv) cells. In mouse cells, TERF1 was used as a telomere marker. In human cells, TERF2 antibody was used as the telomere marker as the TERF1 antibody did not work in human cell types. Scalebars represent 5μm.

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal antisera against mouse TERF1 ( ); mouse monoclonal antisera against AURKB (BD Transduction Laboratories, #611082); mouse monoclonal antisera against GFP (Roche, #11814460001), rabbit polyclonal antisera against phosphorylated H3.3 serine 31 (Active Motif, #39637), mouse monoclonal antisera against TERF2 (Santa Cruz, #sc-47693) and rat monoclonal antisera against hemagglutinin (HA) tag (Roche, #11867423001).

Techniques: Marker

Journal: Nucleic Acids Research

Article Title: Aurora Kinase B, a novel regulator of TERF1 binding and telomeric integrity

doi: 10.1093/nar/gkx904

Figure Lengend Snippet: Loss of AURKB activity in ESCs results in the formation of MTS. ( A ) Examples of MTS (obtained with APH treatment) shown. ( B ) Representative metaphase images of untreated control mouse ES129.1 cells (i) and those treated with either 0.2 µM APH (ii) or 1 µM AURKB inhibitor ZM447439 (iii) for 24 h. TEL-FISH analyses indicated that 24 h of 1 µM ZM447439 treatment resulted in an increase in MTS formation from an average of 2.3 of MTS/metaphase in untreated control cells to 8.5 MTS/metaphase in ZM447439 treated cells ( P < 0.0001; N = 1000 chromosomes from three biological replicates), compared to an average of 7.3 MTS/metaphase in cells treated with 0.2 µM APH ( P < 0.0001; N = 1000 chromosomes from three biological replicates) (iv and v). ( C ) Western blot analyses of AURKB and actin in ES129.1 cells subjected to scramble control siRNA and siRNA depletion of TERF1, TERF2 and AURKB, respectively (i). Representative images of metaphase ES129.1 cells subjected to scramble control siRNA (ii; negative control), 72 h of AURKB (iii) and TERF1 siRNA depletion (iv), respectively. About 72 h of AURKB depletion resulted in aberrant MTS formation, increasing from an average of 2.3 MTS/metaphase in cells subjected to scramble control siRNA depletion to 5.1 MTS/metaphase in AURKB-depleted cells ( P = 0.0006, N = 1200 chromosomes from three biological replicates) (v and vi). As a comparison, 72 h of TERF1 siRNA depletion caused an average of 19.95 MTS/metaphase ( P < 0.0001; Cv and vi). Magnified images of the boxed chromosomes in B and C are shown in the inset, with examples of MTS indicated by the arrowheads. Each point in scatterplots (Biv and Cv) represents of the number of MTS in a single metaphase spread, with error bars showing Q1, Q2 and Q3 values. P -values are indicated in column graphs (Biv and Cv). Scalebars represent 5 μm.

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal antisera against mouse TERF1 ( ); mouse monoclonal antisera against AURKB (BD Transduction Laboratories, #611082); mouse monoclonal antisera against GFP (Roche, #11814460001), rabbit polyclonal antisera against phosphorylated H3.3 serine 31 (Active Motif, #39637), mouse monoclonal antisera against TERF2 (Santa Cruz, #sc-47693) and rat monoclonal antisera against hemagglutinin (HA) tag (Roche, #11867423001).

Techniques: Activity Assay, Control, Western Blot, Negative Control, Comparison

Journal: Nature structural & molecular biology

Article Title: The macroH2A1.2 histone variant links ATRX loss to alternative telomere lengthening.

doi: 10.1038/s41594-019-0192-3

Figure Lengend Snippet: (A) ChIP for γ-H2AX at the indicated loci in K562 cells in the presence (WT) or absence of macroH2A1.2 (1.2 CRISPR-KO). Samples were normalized to untreated WT samples, values represent mean and s.d. from 3 independent experiments. See for macroH2A1.2 ChIP. (B) γ-H2AX ChIP at the indicated loci in U2OS cells treated with vehicle (DMSO) or HU in the presence or absence of ATRX re-expression. Values represent mean and s.d. from 3 independent experiments. (C) Frequency of TIFs in U2OS cells with ATRX induction in the presence or absence of Aph, TIFs were defined based on co-localization of γ-H2AX (red) and TRF2 (green), a representative image from Aph-treated, sh-RFP expressing U2OS cells is shown; scale bar = 5 μm. Box plots depict the number of TIFs per cell. N: number of cells. *** p < 10 −8 by Mann-Whitney U test, one of two independent experiments is shown. (D) Model linking ATRX and macroH2A1.2 to ALT telomere maintenance. ATRX-dependent macroH2A1.2 retention at stalled replication forks protects from excessive DNA damage in ALT-negative cells. In ALT-positive cells, ATRX deficiency leads to macroH2A1.2 loss and DSBs in response to RS, which triggers DDR-dependent macroH2A1.2 re-deposition to facilitate HR. In the absence of RS, lack of ATRX has little effect on telomeric macroH2A1.2 levels, pointing to DNA damage-induced modulation of ATRX function, which may involve ATRX phosphorylation in S phase (see ) .

Article Snippet: The following antibodies were used for ChIP: α-macroH2A1.2 (Millipore MABE61), α-phospho-S139-H2AX (Millipore 05–636), α-H2B (Abcam ab52484), α-H2A (Abcam ab18255), TRF2 (Novus Biologicals IMG-124A) and normal mouse IgG (Millipore 12–371).

Techniques: CRISPR, Expressing, MANN-WHITNEY, Phospho-proteomics

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