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