trf2  (Novus Biologicals)

Journal: Cell reports

Article Title: Local heterochromatin enrichment promotes telomere clustering and PML nuclear body assembly at telomeres

doi: 10.1016/j.celrep.2026.117004

Figure Lengend Snippet: (A) Schematic of the fusion-protein constructs. Top: full constructs contain a TetON doxycycline (DOX)-inducible promoter, a Myc-tagged-TRF1 fused to a chromatin modifier (here, KRAB), a T2A, and mCherry-NES (nuclear export signal). (B) Western blot showing expression of the constructs (anti-Myc antibody) and H3K9me3 global levels in U2OS after 3 days of DOX induction. (C) Representative chromatin immunoprecipitation of H3, H3K9me3, and HP1α at telomeres and ALU repeats in U2OS after 3 days of DOX induction (T-KRAB: TRF1-KRAB). (D) Quantification of (C). Data represent mean ± SEM of n = 3 independent biological replicates. (E) Representative images of IF-FISH assessing PML colocalization with telomeres (TTAGGG) in U2OS after 3 days of DOX induction. Scale bars, 10 μm. (F–I) Quantification of (E). (F) Number of telomere foci per cell. (G) Standard deviation of the telomere foci area within each cell. (H) Number of total (small and large) telomere-PML colocalizations (TPF). (I) percentage of cells with at least 5 APBs. (F–H) Data represent mean ± SEM of all cells analyzed over three biological replicates. n (number of cells analyzed) = 110 (WT), 115 (TRF1), and 113 (TRF1-KRAB). (I) Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. (J) Representative C-circle assay in U2OS after 3 days of DOX induction. Right: quantification. Data represent mean ± SEM of n = 3 independent biological replicates. (K) G2 telomere synthesis: colocalization of TRF2 with G2-incorporated EdU in U2OS after 3 days of DOX induction. Scale bars, 10 μm. Right: quantification. Data represent mean ± SEM of 3 independent biological replicates, with at least 30 cells analyzed per replicate. Statistical analyses: for (D and F–I), ordinary one-way ANOVA. For (J and K), Welch’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns, non-significant. See also .

Article Snippet: Coverslips were stained for rabbit- anti -TRF2 (Novus, #NB110-57130 dilution 1:200) immunofluorescence as described above.

Techniques: Construct, Western Blot, Expressing, Chromatin Immunoprecipitation, Standard Deviation

Journal: Cell reports

Article Title: Local heterochromatin enrichment promotes telomere clustering and PML nuclear body assembly at telomeres

doi: 10.1016/j.celrep.2026.117004

Figure Lengend Snippet: (A) Schematic of the fusion-protein constructs. Here, Myc-TRF1 is fused to HP1α V22M, a mutation that abrogates H3K9me3 binding. (B) Western blot showing the expression of the constructs (anti-Myc antibody) in U2OS after 3 days of DOX induction. (C)Representative chromatin immunoprecipitation of H3, H3K9me3, and HP1α at telomeres and ALU repeats in U2OS after 3 days of DOX induction (T-HP1α: TRF1-HP1α). (D) Quantification of (C). Data represent mean ± SEM of n = 3 independent biological replicates. (E) Representative images of IF-FISH assessing PML colocalization with telomeres (TTAGGG) in U2OS after 3 days of DOX induction. Scale bars, 10 μm. (F) Quantification of cells with at least 5 APBs from (E). Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. (G) Representative C-circle assay in U2OS after 3 days of DOX induction. Right: quantification. Data represent mean ± SEM of n = 3 independent biological replicates. (H) G2 telomere synthesis: colocalization of TRF2 with G2-incorporated EdU in U2OS after 3 days of DOX induction. Left: representative images. Scale bars, 10 μm. Right: quantification. Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. (I) Quantification from (E) of the standard deviation between telomeric foci area within each cell. Data represent mean ± SEM of all cells from 3 independent biological replicates. n (number of cells analyzed) = 110 (WT), 115 (TRF1), and 100 (TRF1-HP1α). (J) Representative image from experiment in (E) of a cell with ultra-bright APBs (left) and cells with a telomeric bridge (right) in U2OS + TRF1-HP1α. Scale bar: 10 μm. (K and L) Percentage of cells with ultra-bright APBs (K) and entanglements (L). Data represent mean ± SEM of 3 independent biological replicates, with at least 30 cells analyzed per replicate. Statistical analyses: for (D, F, I, and K–L), ordinary one-way ANOVA. For (G and H), Welch’s t test. * p < 0.05, ** p < 0.01, and ** p < 0.0001. ns, non-significant. See also .

Article Snippet: Coverslips were stained for rabbit- anti -TRF2 (Novus, #NB110-57130 dilution 1:200) immunofluorescence as described above.

Techniques: Construct, Mutagenesis, Binding Assay, Western Blot, Expressing, Chromatin Immunoprecipitation, Standard Deviation

Journal: Cell reports

Article Title: Local heterochromatin enrichment promotes telomere clustering and PML nuclear body assembly at telomeres

doi: 10.1016/j.celrep.2026.117004

Figure Lengend Snippet: (A) Quantification of basal levels of telomere-PML colocalization (TPF) in HeLa ST and HeLa LT. Data represent mean ± SEM of 3 independent biological replicates. (B) Western blot showing siRNA-mediated knockdown of HP1α in HeLa LT control cells and expressing TRF1-KRAB (T-KRAB). (C) Western blot showing Myc (TRF1-KRAB), HP1α, H3K9me3, and actin in indicated cell lines. (D) Quantification of TPF in indicated cell lines. Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. (E) Representative images of IF-FISH assessing PML (IF) colocalization with telomeres (TTAGGG) in HeLa LT with indicated constructs after 3 days of DOX induction. Scale bars, 10 μm. (F) Quantification of TPF from (E). Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. (G) C-circle assay in HeLa LT after 3 days of DOX induction. Data represent mean ± SEM of n = 3 independent biological replicates. (H) G2 telomere synthesis: colocalization of TRF2 with G2-incorporated EdU in HeLa LT after 3 days of DOX induction. Scale bars, 10 μm. (I) Quantification of (H). Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. (J) Representative image and quantification of entanglements in HeLa LT with indicated constructs. Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. Scale bar: 10 μm. (K) Representative images of IF-FISH assessing PML (IF) colocalization with telomeres (TTAGGG) in HeLa ST with indicated constructs after 3 days of DOX induction. Scale bars, 10 μm. (L and M) Quantification of TPF (L) and entanglements (M) from (K). Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. Statistical analyses: for (B, D, F, I, and L), ordinary one-way ANOVA. For (G, J, and M), Welch’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns, non-significant. See also .

Article Snippet: Coverslips were stained for rabbit- anti -TRF2 (Novus, #NB110-57130 dilution 1:200) immunofluorescence as described above.

Techniques: Western Blot, Knockdown, Control, Expressing, Construct

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-17A Orchestrates Lung Injury and Remodeling Through p53 and uPA System Crosstalk

doi: 10.3390/ijms27041841

Figure Lengend Snippet: Regulation of BLM-LI and TSE-LI by IL-17A. ( A ) Lung sections of WT mice exposed to saline or BLM for 24–72 h were subjected to IHC staining for IL-17A antigen ( i ). AECs isolated from WT and IL-17A-deficient mice exposed to saline or BLM were analyzed for apoptosis and Sirt1 expression by Western blotting (WB) three days later ( ii ). Lung sections of WT and IL-17A −/− mice exposed to saline or BLM were subjected to Masson’s trichrome staining 21 days later ( iii ). Whole lung homogenates of WT and IL-17A −/− mice exposed to saline or BLM as in were analyzed for total HYP content ( iv ) or for Col-1 and FN by WB 21 days after BLM ( v ). ( B ) WT, p53 −/− and PAI-1 −/− mice were kept in ambient air or exposed to passive TS for 20 weeks. Total lung homogenates of control mice kept in ambient air, TSE WT mice, and p53 −/− or PAI-1 −/− mice were analyzed for IL-17A and IL-17RA protein by WB ( i ). Total RNA from mice kept in ambient air, TSE WT mice, and p53 −/− or PAI-1 −/− mice was analyzed for IL-17A mRNA ( ii ). AECs from TSE WT and IL-17A −/− mice were analyzed for p53, ACp53, cleaved (Cl.) caspase-3/caspase-3 and Sirt1 by WB ( iii ). AECs from lungs of IL-17A −/− mice kept in ambient air or TSE, as well as mice treated with or without CSP7 or CP, were analyzed for telomere length by qPCR ( iv ). AECs from lungs of IL-17A −/− mice treated as in ( B(iv )) were immunoblotted for the listed proteins ( v ). Lung sections of IL-17A −/− mice treated as in ( B ( iv )) were subjected to IHC for telomere binding shelterin complex (TRF1 and TRF2) proteins ( vi ).

Article Snippet: 12 , TRF2 (D1Y5D) rabbit mAb , CST , 13136S , 1:1000 , 1:500.

Techniques: Saline, Immunohistochemistry, Isolation, Expressing, Western Blot, Staining, Control, Binding Assay

Journal: bioRxiv

Article Title: The glycine-arginine-rich motif of 53BP1 modulates RNA interactions necessary for its liquid-liquid phase separation during DNA Damage Response

doi: 10.64898/2026.01.30.702603

Figure Lengend Snippet: (a) Immunoblot for 53BP1, TRF2 and actin (loading control) in Trf2 F/F 53bp1 −/− MEFs expressing empty vector (EV), full-length wild-type 53BP1 (WT) or its GAR mutants (R→A or R→K), before or 96 h after Cre-mediated Trf2 deletion with Hit&Run Cre. (b) Representative metaphase spreads in the indicated MEFs at 96 h after Cre-mediated Trf2 deletion. Telomeres were detected with Alexa Fluor 488-OO-(TTAGGG) 3 (green), DNA with DAPI (magenta). Arrows indicate telomere fusions. The boxed regions are enlarged in the right row. Scale bar = 10 µm. (c-d) Quantification of telomere fusions shown in (b) at 96 h (c) or 120 h (d) after Cre-mediated Trf2 deletion. Data are presented as median from 3 independent experiments (10 metaphases each). Each data point represents the percentage of telomeres fused in one metaphase, with different shapes indicating the different replicates. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: The membrane was blocked in 5% milk in PBS containing 0.1% (v/v) Tween-20 (PBS-T), and immunoblotted using the following antibodies: 53BP1 (ab175933, Abcam), TRF2 (13136, Cell Signaling), β-actin (3700, Cell Signaling), followed by goat anti-rabbit (31460, Invitrogen) or anti-mouse (31430, Invitrogen) IgG-HRP secondary antibodies.

Techniques: Western Blot, Control, Expressing, Plasmid Preparation

Journal: Communications Biology

Article Title: BLM and FANCJ role in the response to G-quadruplex-dependent telomeric replicative stress

doi: 10.1038/s42003-025-09367-z

Figure Lengend Snippet: a The diagram shows the workflow on a timeline: on the day after cell seeding, cells were treated with RHPS4 (0.2 and/or 0.5 μM). At 48 h after treatment, cells were again treated with RHPS4 (0.2 and/or 0.5 μM) or harvested and counted. At 72 h after the first treatment, the cell viability was checked, and finally at 96 h after the first treatment, the cells were harvested and processed. b Representative images showing U251MG cells stained by immunofluorescence using anti-BLM (red signals), and anti-TRF1 or anti-TRF2 (green signals) antibodies. Merged images allow visualization of colocalizing dots (yellow signals). Yellow arrows indicate BLM and TRF1-TRF2 colocalizations. c Quantification of the colocalizations between BLM and TRF1 or TRF2 proteins in U251MG cell line upon RHPS4 treatment (0.2 and 0.5 μM). d , e Sensitivity of U251MG and BLM −/− cell lines to RHPS4 concentrations ranging from 0.01 to 2 μM (0.01; 0.125; 0.25; 0.5; 1; 2 μM), evaluated 96 h after the first treatment. Mitomycin C (MMC) was used as a positive control at concentrations of 0.1; 0.5; 1; 2; 5 μg/ml. The Sulforhodamine B (SRB) cytotoxicity assay showed that RHPS4 sensitivity was unchanged in U251MG and BLM −/− (IC50 was 0.56 μM in both cell lines). f Short-term cell proliferation in untreated cells, U251MG and BLM −/− , and in RHPS4-treated cells (0.5 μM) as evaluated 48 and 96 h after the first treatment. g Long-term cell proliferation in U251MG and BLM −/− untreated and RHPS4-treated cells (0.5 μM). Scale bars represent 5 μm. * p < 0.05, ** p < 0.01 (two-way ANOVA; n = 3). Error bars denote the standard deviation of the mean.

Article Snippet: The slides were then incubated in blocking buffer (1% BSA dissolved in 1X PBS [w/v]) and then incubated overnight (ON) at 4 °C with the primary antibodies (rabbit polyclonal anti-BLM (#A300-110A, Bethyl) (1:100); mouse monoclonal anti-TRF1 (4E4 clone, GTX70304, GeneTex) (1:20) or mouse monoclonal anti-TRF2 (9F10 clone, sc-47693, Santa Cruz Biotechnology) (1:100)).

Techniques: Staining, Immunofluorescence, Positive Control, Cytotoxicity Assay, Standard Deviation