anti terf2 ip  (Novus Biologicals)

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Detection of TERRA and hTR by smiFISH in HeLa cells. The percentage of cells, in which at least 1 TERRA-hTR colocalization event is detected, is indicated (mean ± SD; n = 5; 298 cells analyzed). Scale bar, 5 μm. ( B ) Quantification of the number of TERRA-hTR colocalizations per nucleus (mean ± SD; n = 5; 298 cells analyzed). ( C ) Detection of TERRA, hTR, and telomeres by smiFISH/RAP1 IF in HeLa cells (mean ± SD; n = 2; 102 cells analyzed). Scale bar, 5 μm. ( D ) Quantification of the number of TERRA-hTR colocalizations per cell detected at telomeres (TERRA-hTR-RAP1) and outside telomeres (TERRA-hTR w/o RAP1) (mean ± SD; n = 2; 102 cells analyzed). ( E ) Number of hTR-RAP1 colocalizations per cell with and w/o TERRA (mean ± SD; n = 2; 102 cells analyzed). ( F ) Quantification of the number of TERRA-hTR foci at telomeres (TERRA-hTR-RAP1) and outside telomeres (TERRA-hTR w/o RAP1) during G 1 , S, and G 2 phase in HeLa cells upon cell synchronization (mean ± SD; n = 2; total number of cells analyzed: 54 (G 1 phase), 46 (S phase), and 45 (G 2 phase). Fraction of hTR-TERRA foci at telomeres: 20.9 ± 3.3% in G 1 -phase cells, 40.2 ± 11% in S-phase cells, and 41.1 ± 5.5% in G 2 -phase cells.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques:

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Telomere length measurement by TRF through Southern blot in HeLa cells in the indicated conditions. NT, untreated; CTR, DMSO-treated. Estimated average telomere length and elongation rate for each sample are indicated in the table below. Average ± SD from two technical replicates. ( B ) Detection of TERRA and telomeres by smiFISH/IF in CTR, 7PD rescue, and 24PD rescue cells. Scale bar, 5 μm. ( C ) Quantification of the number of telomeric TERRA foci detected per nucleus by smiFISH/IF (each dot represents a nucleus) (mean ± SD; n = 2; 124 CTR cells, 111 7PD rescue cells, and 112 24PD rescue cells analyzed). Unpaired nonparametric Kruskal-Wallis test coupled with post hoc Dunn’s multiple-comparison test: ** P < 0.01, *** P < 0.001. ( D and E ) Distribution analyses of the number of telomeric TERRA foci per cell. Two-way analysis of variance (ANOVA) test: ** P < 0.01. Post hoc Tukey’s multiple-comparison test: group 1 to 4 P values = 0.07 (CTR versus 7PD rescue), 0.04 (7PD rescue versus 24PD rescue), not significant (ns) (CTR versus 24PD rescue). ( F ) Detection of TERRA, hTR, and telomeres by smiFISH/IF in CTR, 7PD rescue, and 24PD rescue cells. Scale bar, 5 μm. ( G ) Quantification of the percentage of TERRA-hTR foci colocalizing at telomeres and extratelomeric (mean ± SD; n = 2; 124 CTR cells, 111 7PDs rescue cells, and 112 24PDs rescue cells analyzed). Two-way ANOVA test: **** P < 0.0001. ( H ) Distribution analyses of the number of TERRA-hTR-RAP1 colocalizing foci per nucleus. Two-way ANOVA test: **** P < 0.0001; multiple-comparison test: P = 0.0003 for 7PD versus 24PD rescue, P < 0.0001 for CTR versus 7PD rescue, P = 0.001 CTR versus 24PD rescue. ( I ) Number of telomeric hTR foci not colocalizing with TERRA per cell detected by smiFISH/IF. Kruskal-Wallis test: P = 0.0025.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Southern Blot, Comparison

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Detection of TERRA and RAP1 by smiFISH/IF in HeLa cells. An example of a colocalization event between TERRA and RAP1 is shown in the image magnifications. DAPI is used to stain nuclei. Scale bar, 5 μm. ( B ) Integrated density quantification of RAP1 foci colocalizing and not colocalizing with TERRA foci in HeLa cells as detected by smiFISH/IF. Each dot represents a single RAP1 focus. Mean ± SD is shown. A total of 110 cells were analyzed in three independent biological replicates. Statistical significance was assessed by Mann-Whitney test. **** P < 0.0001. ( C ) Integrated density quantification of RAP1 foci colocalizing and not colocalizing with TERRA foci in the indicated samples. Each dot represents a single RAP1 focus. Mean ± SD is shown from the following number of samples and biological replicates: 64 CTR cells ( n = 2), 67 BIBR1532 cells (123PDs of treatment with BIBR 1532) ( n = 2), 111 7PD rescue cells ( n = 2), 151 POT1 WT cells ( n = 3), and 148 POT1-ΔOB cells ( n = 3). The Mann-Whitney test was used to assess statistical significance. **** P < 0.0001.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Staining, MANN-WHITNEY

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Detection of TERRA and telomeres in HeLa cells by smiFISH/IF. Image acquisitions were performed using three-dimensional structured illumination microscopy (3D-SIM). Examples of TERRA foci colocalizing with a single telomere (top images) or telomere doublet (bottom images) are displayed. TERRA is shown in red; telomeres are in green. Scale bar is indicated in the rotated view images. ( B ) Quantification of the fraction of single telomeres versus telomere doublets colocalizing with TERRA. Data are shown as percentage of TERRA-colocalizing telomeres and represent mean ± SD from three independent biological replicates for a total of 30 cells and 663 TERRA-colocalizing RAP1 foci analyzed. ( C and D ) Quantification of the integrated density (C) and volume (D) of RAP1 foci colocalizing (with TERRA) and not colocalizing (without TERRA) with TERRA. Both TERRA-single telomere and TERRA-telomere doublet colocalizations were considered. Data are shown as arbitrary units (a.u.), in (C), and μm 3 , in (D), and represents mean ± SD from three independent biological replicates for a total of 30 cells and 663 TERRA-colocalizing RAP1 foci analyzed. The Mann-Whitney test was used to assess statistical significance. **** P < 0.0001. ( E ) Quantification of the integrated density of all TRF1-mCherry foci and TRF1-mCherry foci colocalizing with MS2-tagged telomere 15q TERRA transcripts per nucleus. Forty nuclei corresponding to 3690 telomeres and 73 telomeres colocalizing with MS2-TERRA transcripts were analyzed from imaging datasets obtained in . Statistical analysis was performed with a Kolmogorov-Smirnov test: P ≤ 0.0001.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Microscopy, MANN-WHITNEY, Imaging

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Northern blot analysis of TERRA in HeLa cells upon TERRA-ASO or control ASO (ASO SCR) transfection. Bottom image shows 18 S rRNA band upon gel run. ( B ) Quantification of TERRA signal from Northern blot analyses of TERRA-ASO–transfected cells shown as fold over ASO SCR (dashed line). * P < 0.05; mean ± SD, n = 2. ( C ) RT-qPCR analyses of TERRA expression from the indicated telomeres in TERRA-ASO cells shown as fold over ASO SCR. Mean ± SD from four independent biological replicates. Unpaired t test: ** P < 0.01, *** P < 0.001. ( D ) Integrated density quantification of TERRA foci colocalizing with RAP1 foci. Each dot represents a single TERRA signal (mean ± SD; n = 2; 131 ASO SCR cells and 122 TERRA-ASO cells analyzed). Mann-Whitney test: ** P < 0.01. ( E ) Quantification of the number of hTR foci detected per nucleus in TERRA-ASO and ASO SCR cells (mean ± SD; n = 2; 131 ASO SCR cells and 122 TERRA-ASO cells analyzed). Mann-Whitney test: **** P < 0.0001. ( F ) RT-qPCR quantification of hTR levels using primer pairs detecting the precursor or mature RNA ( , ). Results are shown as fold change over ASO SCR (dashed line) (mean ± SD, n = 2). U6 gene was used for normalization . ( G ) Detection of hTR and telomeres by smiFISH/IF. Scale bar, 5 μm. ( H ) Quantification of the number of telomeric hTR foci detected per nucleus. Data are shown as number of RAP1-hTR colocalizations per cell (each dot represents a cell) (mean ± SD, n = 2; 131 ASO SCR cells and 122 TERRA-ASO cells analyzed). Mann-Whitney test: **** P < 0.0001. ( I and J ) Distribution analysis of the number of RAP1-hTR colocalizations detected per cell. Two-way ANOVA test: ** P < 0.01.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Northern Blot, Control, Transfection, Quantitative RT-PCR, Expressing, MANN-WHITNEY