Journal: Nature Communications

Article Title: A CPC-shelterin-BTR axis regulates mitotic telomere deprotection

doi: 10.1038/s41467-025-57456-8

Figure Lengend Snippet: a TRF2 domain structure and the mutant alleles used in this study. Predicted AURKB sites in the basic domain are indicated. b Below, representative immunoblots of Myc immuno-precipitates from TRF2 shRNA HT1080 6TG expressing Myc-TRF2-WT. Cells were synchronized with a thymidine block, released in the presence or absence of 150 ng mL −1 nocodazole (Noc) and the indicated kinase inhibitors for 16 h. Above, quantitation of normalized anti-TRF2-pS65 immunoblot signal (mean +/- s.e.m., n = 3 biological replicates). c Left, example of anti-TRF2-pS65 immunoblot measuring in vitro AURKB kinase assay on purified Myc-TRF2. Right, quantitation of anti-TRF2-pS65 intensity and the resulting Km (mean +/- s.e.m., n = 3 biological replicates, Km is 1.3 (95% CI: 0.49–4.0) fmol). d , e Quantitation of interphase-telomere deprotection induced foci (TIF) ( d ) and metaphase-TIF ( e ) in TRF2 shRNA IMR90 E6E7 hTERT expressing TRF2-WT or the indicated TRF2 alleles. For ( d ) Above, all data points from n = 3 biological replicates of 45 nuclei per replicate compiled into a Tukey boxplot, Kruskal-Wallis followed by Dunn’s multiple comparisons test. Below, representative immunoblots of whole-cell extracts derived from the cell cultures used in this experiment. For ( e ), cells were treated with 2 or 24 h of 100 ng mL −1 colcemid (all data points from n = 3 biological replicates of 15 and 30 metaphases per replicate for 2 h and 24 h colcemid, respectively, compiled into a Tukey boxplot, Kruskal-Wallis followed by Dunn’s multiple comparisons test). Source data are provided as a file.

Article Snippet: Polyclonal rabbit antibodies against phopsho-TRF1 and TRF2 were generated by Genscript.

Techniques: Mutagenesis, Western Blot, shRNA, Expressing, Blocking Assay, Quantitation Assay, In Vitro, Kinase Assay, Purification, Derivative Assay

Journal: Nature Communications

Article Title: A CPC-shelterin-BTR axis regulates mitotic telomere deprotection

doi: 10.1038/s41467-025-57456-8

Figure Lengend Snippet: a , b Interphase-telomere deprotection induced foci (TIF) ( a ) and Metaphase-TIF ( b ) in Trf2 F/F Cre-ER LgT MEFs expressing TRF2-WT or the indicated TRF2 alleles. Where indicated, endogenous mTrf2 was deleted by 4-Hydroxytamoxifen (4-OHT) addition 36 h prior to sample fixation. In ( b ) cells were treated with 14 h of 400 ng mL −1 Nocodazole prior to sample preparation (all data points from n = 3 biological replicates of ≥ 50 cells ( a ) or 30 metaphases ( b ) per replicate, compiled into a Tukey box plot, Kruskal-Wallis followed by Dunn’s multiple comparisons test). c Representative examples of telomere macromolecular structure as visualized by AiryScan microscopy from Trf2 F/F Cre-ER LgT MEFs treated with 400 ng mL −1 Nocodazole for 14 h and collected by mitotic shake-off. Samples were trioxsalen cross-linked in situ, and the chromatin spread on coverslips through cytocentrifugation before telomere FISH labelling . Scale bar, 2 µm. Representative of n = 3 biological replicates. d Quantification of looped telomeres from Trf2 F/F Cre-ER LgT MEFs expressing the indicated TRF2 alleles in mitotically arrested samples prepared and shown as in ( c ) and Supplementary Fig. . Where indicated, endogenous mTrf2 was deleted by 4-OHT addition 36 hours prior to sample fixation (mean +/- s.e.m., n = 3 biological replicates of 200 telomeres per replicate, Ordinary one-way ANOVA followed by Šídák’s multiple comparisons test, F = 6.463, DF = (6, 14)). Source data are provided as a Source Data file.

Article Snippet: Polyclonal rabbit antibodies against phopsho-TRF1 and TRF2 were generated by Genscript.

Techniques: Expressing, Sample Prep, Microscopy, In Situ

Journal: Nature Communications

Article Title: A CPC-shelterin-BTR axis regulates mitotic telomere deprotection

doi: 10.1038/s41467-025-57456-8

Figure Lengend Snippet: a BLM domain structure and the mutant alleles used in this study. The compromised function of the mutant alleles is indicated. b , c Above, Metaphase-telomere deprotection induced foci (TIF) following 24 h of 100 ng mL −1 colcemid in IMR90 E6E7 hTERT expressing Control or BLM shRNA and shRNA-resistant BLM alleles (all data points from n = 3 biological replicates of 30 metaphases per replicate, compiled into a Tukey Box Plot, Kruskal-Wallis followed by Dunn’s multiple comparisons test). Below: representative immunoblots of whole cell extracts derived from the untreated cell cultures used in this experiment. d TOP3A domain structure and the mutant allele used in this study. e Above, Metaphase-TIF following 24 h of 100 ng mL −1 colcemid in IMR90 E6E7 hTERT fibroblasts expressing Control or TOP3A shRNA and vector or shRNA-resistant TOP3A alleles (all data points from n = 3 biological replicates of 30 metaphases per replicate compiled into a Tukey Box Plot, Kruskal-Wallis followed by Dunn’s multiple comparisons test). Below, representative immunoblots of whole cell extracts derived from the untreated cell cultures used in this experiment. f Above, Metaphase-TIF following 24 h of 100 ng mL −1 colcemid in IMR90 E6E7 hTERT expressing Control, BLM or TRF2 shRNA, and vector or TRF2-2D (S62D and S65D) (all data points from n = 3 biological replicates of 30 metaphases per replicate compiled into a Tukey Box Plot, Kruskal-Wallis followed by Dunn’s multiple comparisons test). Below, representative immunoblots of whole cell extracts derived from the untreated cell cultures used in this experiment. Source data are provided as a file.

Article Snippet: Polyclonal rabbit antibodies against phopsho-TRF1 and TRF2 were generated by Genscript.

Techniques: Mutagenesis, Expressing, Control, shRNA, Western Blot, Derivative Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: A CPC-shelterin-BTR axis regulates mitotic telomere deprotection

doi: 10.1038/s41467-025-57456-8

Figure Lengend Snippet: a Mitotic duration for TRF2 shRNA IMR90 E6E7 hTERT expressing the indicated TRF2 alleles. Cells were observed for 50 h in cultures treated with 100 ng mL −1 colcemid (median of N cells from n = 2 experimental replicates). b Representative captures from phase contrast live imaging in ( a ). Mitotic outcomes are indicated. Time is shown as hours: minutes relative to mitotic entry. Scale bar, 20 µm. Representative of n = 2 experimental replicates. c Ratio of mitotic outcomes as shown in ( b ) after indicated duration of mitotic arrest ( N cells from n = 2 experimental replicates). d Number of mitotic cell death and mitotic slippage from ( c ) in the indicated conditions within 2–6 h of mitotic arrest ( n = 2 experimental replicates, unpaired two-tailed Fisher’s Exact test). e Model of MAD telomere deprotection. (1) AURKB phosphorylates TRF1 at S354 and T358; (2) Survivin interacts with pS354 and pT358 to promote (3) AURKB phosphorylation of TRF2 at S62 and S65, enabling (4) BTR to dissolve t-loops. Source data are provided as a file.

Article Snippet: Polyclonal rabbit antibodies against phopsho-TRF1 and TRF2 were generated by Genscript.

Techniques: shRNA, Expressing, Imaging, Two Tailed Test, Phospho-proteomics