mouse anti trf2 img 124a  (Novus Biologicals)

Journal: Cell

Article Title: Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres

doi: 10.1016/j.cell.2016.07.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-TRF2 mouse monoclonal , Imgenex , IMG-124A.

Techniques: Recombinant, Sequencing, Software

Journal: Cell

Article Title: Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres

doi: 10.1016/j.cell.2016.07.033

Figure Lengend Snippet: (A) Summary of the doubly genome-edited cell line generated to track telomeres and single telomerase RNPs and to visualize Cajal bodies. TRF2 and TERT were fused N-terminally with HA-mEos3.2 tag and FLAG-HaloTag, respectively. BFP-coilin was transiently expressed. (B) Western blot and fluorescence imaging of TERT immuno-purified from parental HeLa and three genome-edited cells lines, using FLAG and TERT antibodies (* tagged TERT, # endogenous TERT). The HaloTag and SNAP-tag were labeled with JF646. (C) Direct telomerase extension assay after immuno-purification of TERT. LC1 and LC2, labeled DNA loading controls. (D) Western blot of TERT purifications used for the telomerase assay shown in panel C (* tagged TERT, # endogenous TERT). (E) Cyto-localization of FLAG-HaloTag-TERT in fixed cells synchronized in S-phase. TERT was labeled with JF646; Cajal bodies and telomeres were stained with antibodies against coilin and TRF2, respectively (white arrows indicate co-localizations). See also Figures S1 and S2.

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature and incubated in blocking buffer (3% BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) or the HA epitope (Abcam, ab18181, 1:1000) and coilin (Santa Cruz, sc-32860, 1:100) or 53BP1 (Novus Biologicals, NB100–304, 1:1000) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies and Abcam, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong ® Diamond Antifade Mountant (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}} P36970 ).

Techniques: Generated, Western Blot, Fluorescence, Imaging, Purification, Labeling, Telomerase Assay, Staining

Journal: Cell

Article Title: Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres

doi: 10.1016/j.cell.2016.07.033

Figure Lengend Snippet: (A) Cyto-localization of K78E FLAG-HaloTag-TERT in fixed cells synchronized in S-phase. TERT was labeled with JF646; Cajal bodies and telomeres were stained with antibodies against coilin and TRF2, respectively. K78E TERT associates with Cajal bodies but not telomeres. (B–D) Comparison of the survival probabilities of stationary wild-type and K78E telomerase particles at different nuclear locations: (B) K78E telomerase particles at telomeres, Cajal bodies, and other nuclear sites, (C) Wild-type and K78E TERT at telomeres, (D) Wild-type and K78E TERT at Cajal bodies and other nuclear sites (N = 18 cells for wild-type, N = 14 cells for K78E TERT). K78E TERT particles at telomeres have dynamics indistinguishable from those at other nuclear locations, and they have a reduced survival probability compared to wild-type TERT. In contrast, survival probabilities at Cajal bodies and other nuclear locations are identical for both TERT proteins (also see Fig. S3F–H). (E) Diffusion coefficient histogram of telomeric K78E telomerase tracks present for at least 5 consecutive frames (N=14 cells). K78E telomerase lacks the highly static population (D ~ 0.001 μm2/s). See also Figure S3 and Movie S8.

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature and incubated in blocking buffer (3% BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) or the HA epitope (Abcam, ab18181, 1:1000) and coilin (Santa Cruz, sc-32860, 1:100) or 53BP1 (Novus Biologicals, NB100–304, 1:1000) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies and Abcam, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong ® Diamond Antifade Mountant (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}} P36970 ).

Techniques: Labeling, Staining, Comparison, Diffusion-based Assay

Journal: Cell

Article Title: Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres

doi: 10.1016/j.cell.2016.07.033

Figure Lengend Snippet: (A) Diagram illustrating our approach for visualizing Cajal bodies, telomerase, and telomeres. First, BFP-coilin was imaged, also converting mEOS-3.2 from the green to the red state. Immediately afterwards, we simultaneously imaged red mEOS3.2-TRF2 and HaloTag(JF646)-TERT at 45 frames per second. (B) Still images from movies simultaneously visualizing telomeres (mEOS3.2) and telomerase (HaloTag-JF646), after imaging Cajal bodies marked by BFP-coilin (white arrows indicate co-localizations). (C) A subset of trajectories of telomerase particles, present for at least 10 frames (~220 ms), generated by single-particle tracking of telomerase signals at 45 fps (20 ms exposure), demonstrating rapid three-dimensional diffusion. (D) All TERT trajectories detected in a 45 s movie. Unexplored region marked by asterisk. (E) Diffusion coefficient histogram of telomerase tracks present for at least 5 consecutive frames (N = 18 cells, n = 5035 tracks). Two freely diffusing populations (D ~ 0.3 μm2/s, magenta; D ~ 1.3 μm2/s, blue) and a smaller less mobile population (D ~ 0.01 μm2/s, green) are present. Fractions of the total number of particles in each population are indicated. Also see Movies S1–S4.

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature and incubated in blocking buffer (3% BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) or the HA epitope (Abcam, ab18181, 1:1000) and coilin (Santa Cruz, sc-32860, 1:100) or 53BP1 (Novus Biologicals, NB100–304, 1:1000) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies and Abcam, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong ® Diamond Antifade Mountant (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}} P36970 ).

Techniques: Imaging, Generated, Single-particle Tracking, Diffusion-based Assay

Journal: Cell

Article Title: Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres

doi: 10.1016/j.cell.2016.07.033

Figure Lengend Snippet: Kymographs of telomerase tracks overlapping with TRF2 signals, showing (A) static long-lasting and (B) dynamic “probing” interactions. (C) Approach to analyze static TERT particles. Each TERT track segment represents the distance traveled in a single time interval (~22 ms). Large distances correspond to higher diffusion coefficients. When a binding event occurs, TERT signals remain largely static for several consecutive frames (arrows). By constraining the diffusion coefficient of the tracking algorithm to D < 0.05 μm2/s, only stationary TERT trajectories are analyzed. The length of these tracks corresponds to the time the particle is bound to a particular nuclear locus. (D) Quantification of the survival probability of stationary telomerase particles associated with telomeres, Cajal bodies and nuclear sites (N = 18 cells). TERT particles associate with Cajal bodies and telomeres for longer times than with other nuclear loci. (E) Frequency distribution of the occurrence of stationary particles per telomere in a 45 s timespan (N = 18 cells). (F) Distribution of the duration of long, static interactions between telomerase and telomeres (N = 13 cells, n = 29 interactions, horizontal bar = median, box = 25–75% percentile, whiskers = min-max). Also see Movies S5–S7.

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature and incubated in blocking buffer (3% BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) or the HA epitope (Abcam, ab18181, 1:1000) and coilin (Santa Cruz, sc-32860, 1:100) or 53BP1 (Novus Biologicals, NB100–304, 1:1000) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies and Abcam, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong ® Diamond Antifade Mountant (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}} P36970 ).

Techniques: Diffusion-based Assay, Binding Assay

Journal: Cell

Article Title: Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres

doi: 10.1016/j.cell.2016.07.033

Figure Lengend Snippet: (A) Approach to spatially segregate telomerase tracks. Tracks were assigned to telomeres or Cajal bodies if they came within 3 pixels (0.48 μm) of the centroid of a TRF2 signal or 5 pixels (0.8 μm) of the centroid of a coilin signal, respectively. (B) Diffusion coefficient histograms of telomerase trajectories close to telomeres, Cajal bodies, or neither, present for at least 5 consecutive frames (N = 18 cells). Cajal body associated tracks are enriched for a less mobile population (D ~ 0.01 μm2/s), while telomere associated tracks instead display a highly static population (D ~ 0.001 μm2/s). Fractions of the total number of particles in each population are indicated. (C) Diffusion coefficient histogram of TRF2 tracks. Telomeres move with the same diffusion coefficient as the highly static telomere-associated TERT population (D ~ 0.001 μm2/s).

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature and incubated in blocking buffer (3% BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) or the HA epitope (Abcam, ab18181, 1:1000) and coilin (Santa Cruz, sc-32860, 1:100) or 53BP1 (Novus Biologicals, NB100–304, 1:1000) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies and Abcam, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong ® Diamond Antifade Mountant (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}} P36970 ).

Techniques: Diffusion-based Assay

Journal: Cell

Article Title: Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres

doi: 10.1016/j.cell.2016.07.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature and incubated in blocking buffer (3% BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) or the HA epitope (Abcam, ab18181, 1:1000) and coilin (Santa Cruz, sc-32860, 1:100) or 53BP1 (Novus Biologicals, NB100–304, 1:1000) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies and Abcam, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong ® Diamond Antifade Mountant (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}} P36970 ).

Techniques: Recombinant, Sequencing, Software

Journal: Genome Biology

Article Title: A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression

doi: 10.1186/s13059-015-0791-1

Figure Lengend Snippet: TERT localizes to telomeres in S but not G1 phase of the cell cycle. a IF analysis of fixed HeLa cells expressing FLAG-SNAP-TERT, synchronized in G1 and S phase of the cell cycle (scale bar = 5 μm). Cells expressing FLAG-SNAP-TERT displayed telomere-localized TERT foci in S but not G1 phase of the cell cycle, while parental cells never showed telomere-localized TERT foci. b FACS analysis of the DNA content of cells synchronized in S phase showed a peak between the 2 N and 4 N peaks of asynchronous cells, confirming that they were in S phase. The G1 cell population contained 2 N and 4 N peaks but was depleted for cells with intermediate DNA content. 4 N cells, which failed to release from their mitotic arrest, were easily distinguished from G1 cells by their morphology. c Quantification of the number of TERT foci which co-localized with TRF2 signals in edited HeLa cells synchronized at different stages of the cell cycle. Data were generated from two independent experiments, each analyzing 50 cells per condition (mean ± standard deviation). d FACS analysis of the DNA content of edited HeLa cells released from a double thymidine block as they transition through S phase. Prior to release, the cell population contained mostly cells with 2 N DNA content, which progressively increased as the cells underwent DNA replication. Nine to ten hours after release, DNA replication was complete, as indicated by the majority of cells having 4 N DNA content. e Quantification of the number of TERT foci co-localized with TRF2 signals at different time points during S phase (50 cells per time point, mean ± standard error of the mean; for corresponding images see Fig. S4 in Additional file ). A.U. arbitrary units, Propidium Iodide (PI)

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature, and incubated in blocking buffer (3 % BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) and coilin (Santa Cruz, sc-32860, 1:100) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies, A-31556, and Abcam, ab150117, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong® Diamond Antifade Mountant (Life Technologies, P36970).

Techniques: Expressing, Generated, Standard Deviation, Blocking Assay