Journal: Nature

Article Title: Break induced replication orchestrates resection dependent template switch

doi: 10.1038/s41586-023-06177-3

Figure Lengend Snippet: a. PICh-WB for telomere associated RAD18, SNM1A, PCNA and PCNA-Ub (Ubiquitylated PCNA (Lys164)) in HeLa S3 and U2OS induced with TRF1-FokI (D450A and WT), respectively. GAPDH, TRF2 and mCherry (mCherry tagged TRF1-FokI) were blotted as experimental controls. b-c. Nuclear extracts from U2OS cells or HeLa S3 stably expressing SNM1A (or mutants) following induction with TRF1-FokI for 2 hrs were subjected to immunoprecipitation (IP) with anti-HA antibody. Input and IP samples were separated with SDS-PAGE and blotted with mCherry (for mCherry tagged TRF1-FokI), SNM1A, TRF2, ubiquitinated PCNA, PCNA and H2A (loading control) antibodies. d. Nuclear extracts from HeLa S3 stably expressing SNM1A (or mutants) after treatment with replication stress agents were subjected to immunoprecipitation (IP) with anti-HA antibody. Input and IP samples were separated with SDS-PAGE and blotted with pATR (T1989), SNM1A, TRF2, ubiquitinated PCNA (Ubiquityl PCNA (Lys164)), PCNA and H2A (loading control) antibodies. “LE”: long exposure; “SE”: short exposure. e. Experimental procedure for BrdU-IP (for (f)) or PICh (Fig.2h, ​,i)i) in HeLa S3 cells induced with TRF1-FokI with aphidicolin at the indicated concentrations. f. BrdU pulldown slot blot for telomere (or Alu) content using 32P-labeled telomeric G-probe or Alu probe, from HeLa S3 cells induced with TRF1-FokI for 2 hrs in presence of aphidicolin (Aphi). g. Quantification of (f). Relative abundance of telomere content enriched by BrdU pulldown are normalized to the uninduced sample. Data represent the mean ± SEM of three independent experiments. The uncropped gel and dot blot images are provided in Supplementary Fig. 1.

Article Snippet: The following primary antibodies were used for western blot: ATR (1:1000, sc-515173, Santa Cruz), ATRIP (1:1000, 2737T, Cell Signaling Technology), Phospho RPA32 (S33) (1:1000, A300–246A, Bethyl Laboratories), PCNA (1:1000, 13110S, Cell Signaling Technology), Ubiquityl PCNA (Lys164) (1:1000, 13439S, Cell Signaling Technology), PolD3 (1:1000, H00010714-M01, Abnova), FANCD2 (1:1000, sc-20022, Santa Cruz), Mre11 [12D7] (1:1000, GTX70212, GeneTex), RAD51 (1:1000, 70–001, BioAcademia), KU70 (1:1000, A302–624A, Bethyl Laboratories), XRCC4 (1:1000, sc-365055, Santa Cruz), TRF2 (1:1000, NB110–57130, Novus Biologicals), GAPDH (1:1000, 2118S, Cell Signaling Technology), Monoclonal ANTI-FLAG ® M2 antibody (1:1000, F1804, Mouse), RAD18 (1:1000, 9040S, Cell Signaling Technology), SNM1A (1:1000, A303–747A, Bethyl Laboratories), mCherry (1:1000, ab183628, Abcam), H2A (1:1000, 07–146, EMD Millipore), ExoI (1:1000, A302–640A-T, Bethyl Laboratories), α-Tubulin (1:200, 12G10, Developmental Studies Hybridoma Bank), HA.11 (1:1000, 901514, Biolegend), Phospho ATR (Thr1989) (1:1000, 58014S, Cell Signaling Technology), DNA2 (1:1000, ab96488, Abcam), GFP (D5.1) (1:1000, 2956S, Cell Signaling Technology).

Techniques: Stable Transfection, Expressing, Immunoprecipitation, SDS Page, Control, Dot Blot, Labeling

Journal: Cancer biology & therapy

Article Title: Sp1 upregulates expression of TRF2 and TRF2 inhibition reduces tumorigenesis in human colorectal carcinoma cells.

doi: 10.4161/cbt.8.22.9880

Figure Lengend Snippet: Figure 1. TRF2 overexpression in tissues and cell line of colorectal cancer (A) Immunohistochemical staining of paraffin-embedded colorectal carcinoma tissues and peritumoral normal tissues. (a) colorectal carcinoma tissue; (b) paired normal colorectal tissue; (c) colorectal carcinoma tissue with peritumoral normal colorectal tissues. Left, X50. Right, X200. (B) Semiquantitative RT-PCR analysis of TRF2 expression in colorectal carcinoma tissues (n = 16) and normal tissues (n = 12). ∆Ct was calculated as Ct (actin)-Ct (TRF2). Statistical significance was shown using unpaired t test, *p < 0.05. (C) Western blots analysis of 3 tumor tissues and 4 normal tissues, TRF2 expression was normalized to β-actin. (D) TRF2 is dispersed throughout the nuclei in SW480 tumor cells, while primary normal cells maintain punctuate TRF2 distribution, DNA was stained with DAPI. The above experiments were performed in triplicate. Representative pictures are shown.

Article Snippet: After preincubated, the samples were either directly used in the binding assay, incubated for 10 min at room temperature with 6 pmol (200-fold molar excess) unlabeled competitor probe, or incubated for 60 min at 4°C with mouse monoclonal anti-TRF2 antibody (1:20, Imgenex).

Techniques: Over Expression, Immunohistochemical staining, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

Journal: Cancer biology & therapy

Article Title: Sp1 upregulates expression of TRF2 and TRF2 inhibition reduces tumorigenesis in human colorectal carcinoma cells.

doi: 10.4161/cbt.8.22.9880

Figure Lengend Snippet: Figure 2. Inhibition of anchorage-dependent and anchorage-independent growth of SW480 cells by siRNA targeting TRF2. SW480 cells were transfected with siRNA targeting TRF2 (siRNA), negative control siRNA (NC) or transfection agent (Control). (A) TRF2 expression was detected by Western blots 48 hr post transfection. (B) The anchorage-dependent growth of SW480 cells was inhibited by siRNA targeting TRF2. (C) The anchorage-independent growth of SW480 cells were determined 7 days post transfection. Cells were stained using Giemsa and colonies were counted. Representative pictures are shown in left part. Graph represents means of three experiments in right part.

Article Snippet: After preincubated, the samples were either directly used in the binding assay, incubated for 10 min at room temperature with 6 pmol (200-fold molar excess) unlabeled competitor probe, or incubated for 60 min at 4°C with mouse monoclonal anti-TRF2 antibody (1:20, Imgenex).

Techniques: Inhibition, Transfection, Negative Control, Control, Expressing, Western Blot, Staining

Journal: Cancer biology & therapy

Article Title: Sp1 upregulates expression of TRF2 and TRF2 inhibition reduces tumorigenesis in human colorectal carcinoma cells.

doi: 10.4161/cbt.8.22.9880

Figure Lengend Snippet: Figure 3. siRNA targeting TRF2 increased chromosomal instability and induced apoptosis in SW480 cells. SW480 cells were transfected with siRNA targeting TRF2 (siRNA), negative control siRNA (NC) or transfection agent (Control). (A) Cytofluorimetric analysis of Annexin V-FITC ver- sus PI staining in SW480 cells. Bi-parametric dot plots indicated Annexin V-FITC versus PI staining in TRF2-depleted, negative control and control. The percentages shown in the Annexin V+/PI- and Annexin V+/PI+ regions of each histogram represent the fraction of apoptotic and necrotic cells, respectively. (B) Telomere fusions in metaphase SW480 cells were increased by TRF2 inhibition. End-to-end fusions (one arrow) and ring chromosomes (two arrows) are indicated in left part. The results are summarized in right part showing percentage of cells with 0, 1 and 2 telomeric fusions/total numbers of metaphases in SW480 cells. Results represented the mean values and the standard deviations of three independent experiments.

Article Snippet: After preincubated, the samples were either directly used in the binding assay, incubated for 10 min at room temperature with 6 pmol (200-fold molar excess) unlabeled competitor probe, or incubated for 60 min at 4°C with mouse monoclonal anti-TRF2 antibody (1:20, Imgenex).

Techniques: Transfection, Negative Control, Control, Staining, Inhibition

Journal: Cancer biology & therapy

Article Title: Sp1 upregulates expression of TRF2 and TRF2 inhibition reduces tumorigenesis in human colorectal carcinoma cells.

doi: 10.4161/cbt.8.22.9880

Figure Lengend Snippet: Figure 4. Sp1 regulates TRF2 expression in colon cancer (A) Transient transfection analysis of TRF2 promoter constructs in SW480 cells. Plasmids contain- ing various lengths of the 5'-upstream sequences of the human TRF2 gene were cloned into a luciferase reporter construct and transiently transfected into SW480 cells. Luciferase activity was measured and activity is expressed relative to the internal control (Vector). Data are expressed as mean ± SEM of three experiments (*p < 0.05, compared with vector). 5'-Deletion fragments of the human TRF2 promoter tested were: TRF2 -57, TRF2 -180, TRF2 -480, TRF2 -930. (B) Chip assay confirm Sp1 binding to TRF2 promoter. Antibody against Sp1 and control IgG were used to precipitate protein-DNA complexes. Purified DNA from precipitated protein-DNA complexes were used as template to amplify the fragment containing Sp1 binding sites in TRF2 promoter. (C) SW480 cells were cotransfected with promoter construct pGL3/-180TRF2 and Sp1 expression vector. Luciferase activity was measured (*p < 0.05, com- pared with control). (D) EMSA was performed with nuclear extracts from SW480 cells, protein-DNA complexes were indicated with arrow. In competition binding assays, unlabeled oligonuleotides containing a consensus Sp1 binding sequence was added at 200-fold excess. Supershift analysis was performed with nuclear extracts in the presence of anti-Sp1 antibody. SS, supershift; FP, free probe.

Article Snippet: After preincubated, the samples were either directly used in the binding assay, incubated for 10 min at room temperature with 6 pmol (200-fold molar excess) unlabeled competitor probe, or incubated for 60 min at 4°C with mouse monoclonal anti-TRF2 antibody (1:20, Imgenex).

Techniques: Expressing, Transfection, Construct, Clone Assay, Luciferase, Activity Assay, Control, Plasmid Preparation, Binding Assay, Purification, Sequencing

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: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) Recombinant MBP or MBP-TRF2 were incubated with 32 P-labeled telomeric dsDNA and binding was assessed by EMSA. Excess of unlabeled telomeric and non-telomeric dsDNA were added as competitors. Samples were migrated on non-denaturing acrylamide gel, dried and exposed to X-ray films. B) Recombinant MBP or MBP-TRF2 were incubated with 32 P-labeled non-telomeric dsDNA and binding was assessed by EMSA. C) Recombinant MBP and MBP-TRF2 were coated to the wells of a 96 well-plate and incubated with HaeIII digested DIG-labeled HHV-6A DNA (25 ng/condition) in the presence or absence of competitors. After washing, bound DNA was quantified by adding peroxidase-labeled anti-DIG antibodies and substrate. Results are expressed as mean absorbance +SD of triplicate values. Experiment is representative of two additional experiments. * P<0.001.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Recombinant, Incubation, Labeling, Binding Assay, Acrylamide Gel Assay

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) Schematic representation of the HHV-6A/B genome. The DR6 probe used for hybridization is shown in red. Uninfected and HHV-6A-infected HSB-2 cells (B-D) or uninfected and HHV-6B-infectd Molt-3 cells (E-F) were analyzed for TRF2 binding to viral DNA using ChIP. The input was hybridized with Alu probe to assess quantity of starting material. Anti-IgG (negative control), anti-PolII (positive control) or TRF2 antibodies were used for immunoprecipitation. B) QPCR detection of GAPDH DNA following ChIP. Results are expressed as fold increase over control IgG. C and E) Eluted DNA was hybridized with 32 P-labeled Alu, telomeric (TTAGGG) 3 or HHV-6A (DR6) probes. After hybridization the membranes were washed and exposed to X-ray films. D and F) Densitometric analysis of relative binding of TRF2 to telomeric and viral DNA. Results of one experiment representative of three are presented and are expressed as signal after normalization to input.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Hybridization, Infection, Binding Assay, Negative Control, Positive Control, Immunoprecipitation, Control, Labeling

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: U2OS cells were infected with HHV-6A and analyzed for TRF2 and IE2 expression at 24h, 48h and 72h post-infection by dual color immunofluorescence. A) Representative immunofluorescence of TRF2 and IE2 expression in bystander and IE2 expressing cells at 24, 48h and 72h post infection. B) Mean relative TRF2 expression ± SD in uninfected (blue), IE2- (green-uninfected bystander) or IE2+ (red-infected) cells at 24h, 48h and 72h post infection. Each symbol represents the relative TRF2 expression from a single nucleus.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Infection, Expressing, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) U2OS cells were mock-infected or infected with HHV-6A for 48h after which cells were processed for IF-FISH. Telomeres were labeled in magenta, TRF2 in green and IE2 in red. The panels in the middle row show images of cells with IE2 patches overlapping with large, diffuse TRF2 and telomeric staining (rectangles). The panels in the third row represent infected cells with punctate IE2 pattern colocalizing with TRF2 and telomeres (dashed squares). The colocalization of IE2, TRF2 and telomeres are shown in both 2D and 3D images. B) Uninfected and HHV-6A-infected U2OS cells were transfected with an empty vector, a myc-tagged-TRF1 expression vector. Forty-eight hours later cells were processed for IF-FISH. TRF1 was labeled in green and IE2 in red. Nuclei were stained with DAPI. Images on the far right show 2D colocalization of TRF1 with IE2.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Infection, Labeling, Staining, Transfection, Plasmid Preparation, Expressing

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) A stick diagram of the IE2 protein with various domains identified is presented. B) Colocalization of HHV-6A IE2 protein with telomeres in the absence of viral DNA. U2OS cells were transfected with an empty vector, with IE2 expression vector or with IE2 Δ1290–1500 expression vector. Forty-eight hours later cells were processed for dual color immunofluorescence. Telomeres were labeled in cyan and IE2 in red. Nuclei are outlined by dashed lines. Examples of IE2 colocalizing with telomeres are presented in a 3D view (white arrows). C) The graph represents the mean ± SD % of WT IE2 and Δ1290–1500 IE2 localizing with telomeres. D) Lack of colocalization between HHV-6A p41 and telomeres in uninfected cells. U2OS cells were transfected with an empty vector or with a p41 expression vector. Forty-eight hours later cells were processed for dual color immunofluorescence. TRF2 was labeled green, p41 in red and nuclei outlined by a dashed line.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Transfection, Plasmid Preparation, Expressing, Immunofluorescence, Labeling

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: U2OS cells were transduced with a lentiviral vector coding for a Dox inducible control shRNA (shCtrl) or a shRNA against TRF2 (shTRF2) and selected with puromycin +/- Dox for a week. A) Western blot analysis of TRF2 expression one week post selection. Membranes were also probed with anti-tubulin antibodies to show the input material loaded. B) One week post selection, +Dox cells were infected with HHV-6A for 48h and processed for IFA using anti-TRF2 (green) and anti-IE2 (red). Cells with IE2 in punctate form and cells with large patchy IE2, likely to represent VRC, are shown. Nuclei are outlined by dashed lines. C) The percentage of HHV-6A infected cells (from B) was estimated after counting a minimum of 700 cells and scoring the IE2 + ones. Results are expressed as mean %IE2 + cells ± SD. D) Mean percentage ± SD of IE2 localizing with telomeres in the presence (shCtrl +Dox) and absence (shTRF2 +Dox) of TRF2. Each dot represents the % of IE2 foci localizing with telomeres in one nucleus. ****p<0.0001. E) IF-FISH confocal images of shCtrl (+Dox) and shTRF2 (+Dox) cells analyzed for TRF2 (green), IE2 (red) and telomeres (cyan). Nuclei are outlined by dashed circles. Examples of IE2 localizing with telomeres (top row) or not found with telomeres (bottom row) are highlighted by the dashed polygons.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Transduction, Plasmid Preparation, Control, shRNA, Western Blot, Expressing, Selection, Infection

Journal: PLoS Pathogens

Article Title: Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration

doi: 10.1371/journal.ppat.1008496

Figure Lengend Snippet: A) U2OS cells were transduced with lentiviral vectors expressing a scrambles shRNA (shCtrl) or a shTRF2. After a week of selection, cells were monitored for TRF2 expression by western blot. B) After a week of selection, shCtrl and shTRF2 treated cells were infected with HHV-6A or HHV-6B. After 30 days, DNA was isolated and the relative frequency of integration, relative to shCtrl set at 100%, estimated by ddPCR. *p<0.05.

Article Snippet: The following primary antibody were used: mouse-α-IE2-Alexa-594 [ ], mouse-α-p41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), and mouse-α-Myc (clone 9E10).

Techniques: Transduction, Expressing, shRNA, Selection, Western Blot, Infection, Isolation

Journal: PLoS ONE

Article Title: Human Umbilical Cord Matrix Stem Cells Maintain Multilineage Differentiation Abilities and Do Not Transform during Long-Term Culture

doi: 10.1371/journal.pone.0071374

Figure Lengend Snippet: A. Representative normal G-banded karyotype of cultured UCMSC. B. Relative TRAP activity per microgram of protein lysate. Telomerase activity of UCMSC remained below the positive cut-off represented by heat inactivated (HI: 85°C for 10 min) HeLa cell extracts. C. Quantitative PCR confirmed the absence of hTERT gene transcripts in UCMSC at P9 and P18. HeLa cell cDNA was used as a positive control. D. Representative Southern blot analysis of telomere length. Lane 1 represents molecular weight markers. Telomere length of UCMSC shortened gradually between P3 and P18 (lane 2–4). Control HeLa cells (lane 5) displayed short telomeres, typical of most telomerase-positive cancer cell lines, while U2OS cells (lane 6) displayed the typical long and heterogeneous TRF pattern of ALT-positive cells. E. Cultured UCMSC display signs of telomere dysfunction induced cell senescence. Cells from P3, P9 and P18 were immunostained for 53BP1 DNA damage marker (red) and TRF2 telomeric protein (green). Co-localization of 53BP1 foci with TRF2 was scored on at least 50 nuclei to determine telomere dysfunction induced DNA damage foci (TIF). F. Quantification (%) of nuclei presenting 53BP1 foci at P3, P9 and P18. Percentage of nuclei presenting co-localization of 53BP1 with TRF2 in UCMSC at selected passages. (Scale bar 5 µm).

Article Snippet: Fixed cells were incubated with a mouse anti-human TRF2 primary antibody (1/500; Imgenex, San Diego, USA) and with a rabbit anti-human 53BP1 antibody (1/250; Novus Biologicals, Cambridge, UK) overnight at 4°C.

Techniques: Cell Culture, Activity Assay, Real-time Polymerase Chain Reaction, Positive Control, Southern Blot, Molecular Weight, Control, Marker