2080320 anti gammah2ax ser139 santa cruz cat  (Santa Cruz Biotechnology)

Journal: Nature Communications

Article Title: TRF1 relies on fork reversal to prevent fragility at human telomeres

doi: 10.1038/s41467-025-61828-5

Figure Lengend Snippet: A Above: Representative blot from ChIP analyses showing TRF1 binding at telomeres in PCS ST and PCS LT cells and in PCS LT cells transduced with pLPC empty vector (EV) or pLPC-TRF1 (TRF1). Below: Quantifications. Signals were normalized first to telomeric input signals and then to either PCS LT (left part) or EV-transduced PCS LT cells (right part). n = 3 independent biological replicates for PCS LT and PCS ST and n = 4 independent biological replicates for PCS LT cells transduced with either EV or TRF1. Mean ± SEM. Two-tailed ratio paired t -tests. B Western blot of cell extracts from PCS LT cells transduced with pLPC/pLHCX-empty vector (EV), pLPC-TRF1 (TRF1) or pLHCX-RNaseH1-Myc (RH1), probed for TRF1 and Myc. Representative of n = 3 biological replicates. C Above: Representative CO-FISH pictures on metaphase spreads from PCS LT cells transduced with either EV, TRF1, or both TRF1 and RH1 showing lagging (green) and leading (red) telomere fragility (arrowheads). Below: Lagging (green) and leading (black) telomere fragility. A minimum of 5200 chromosome ends were analyzed, based on n metaphase spreads collected from three independent biological replicates. Boxplot: Min, 1st quartile, Median, 3rd quartile, and Max. Kruskal–Wallis tests. Source data are provided as a Source Data file.

Article Snippet: One hundred microliters of lysate were kept as input and 900 μl were incubated with 1 μg of anti-TRF1 antibody (St John’s laboratory) for 4 h at 4 °C.

Techniques: Binding Assay, Transduction, Plasmid Preparation, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: TRF1 relies on fork reversal to prevent fragility at human telomeres

doi: 10.1038/s41467-025-61828-5

Figure Lengend Snippet: A Above: Western blot of cell extracts from PCS LT cells transduced with pLPC empty vector (EV) or pLPC-TRF1 (TRF1), probed for pRPA32-S33, RPA32, and Actin. Below: pRPA32-S33 (purple) and RPA32 (green) levels, normalized to Actin and to PCS LT -EV. Representative of n = 6 independent biological replicates. Mean ± SEM. Two-tailed ratio paired t -tests. B Co-localization events between telomeres and RAD51 (cyan) or RPA32 (green) in PCS LT cells transduced with either EV or TRF1. n nuclei were analyzed from three independent biological replicates. Median in red, quartiles in orange. Two-tailed Mann–Whitney tests. C Representative blot and quantification for CCA in PCS LT cells transduced with either EV or TRF1. Signals were normalized to PCS LT -EV. n = 3 independent biological replicates. Mean ± SEM. Two-tailed ratio paired t -test. D Co-localization events between telomeres and PML in PCS LT cells transduced with either EV or TRF1. n nuclei were analyzed from three independent biological replicates. Median in red, quartiles in orange. Two-tailed Mann–Whitney test. E Above left: Schematic representation of the pTelN mini-chromosome containing 115 telomeric repeats (red arrow) and the SV40 replication origin (ORI). Above right: Schematic representation of the 2D-gel migration profile of the specified DNA replication intermediates. Below left: 2D-gel analysis of plasmid DNA using the 5.2 kb Bam HI- Sac I probe, in cells with and without ectopic overexpression of TRF1. Below right: Quantification of cone signal (reversed fork migration) as a ratio of cone signal to total replication intermediates (RI), normalized to EV. n = 3 independent biological replicates. Mean ± SEM. Two-tailed ratio paired t -test. F , G Lagging (green) and leading (black) telomere fragility assessed by CO-FISH in PCS LT -EV or PCS LT -TRF1 cells under the specified conditions. siSM1: siSMARCAL1. Cells were treated with either 15 µM BIBR ( F ) or 5 µM Olaparib (PARPi) ( G ), or DMSO as control, during 48 h before BrdU/BrdC incorporation. A minimum of 8600 ( F ) or 6200 ( G ) chromosome ends were analyzed, based on n metaphase spreads collected from three independent biological replicates. Boxplot: Min, 1st quartile, Median, 3rd quartile, and Max. Kruskal–Wallis tests. Source data are provided as a Source Data file.

Article Snippet: One hundred microliters of lysate were kept as input and 900 μl were incubated with 1 μg of anti-TRF1 antibody (St John’s laboratory) for 4 h at 4 °C.

Techniques: Western Blot, Transduction, Plasmid Preparation, Two Tailed Test, MANN-WHITNEY, Two-Dimensional Gel Electrophoresis, Migration, Over Expression, Control

Journal: Nature Communications

Article Title: TRF1 relies on fork reversal to prevent fragility at human telomeres

doi: 10.1038/s41467-025-61828-5

Figure Lengend Snippet: A Lagging (green) and leading (black) telomere fragility assessed by CO-FISH in PCS LT -EV, PCS LT -TRF1 or PCS LT -TRF1-RH1 cells under the specified conditions. siSM1: siSMARCAL1. Cells were treated with 60 µM spironolactone (SP), or DMSO as control, during 25 h before BrdU/BrdC incorporation. A minimum of 5900 chromosome ends were analyzed, based on n metaphase spreads collected from three independent biological replicates. Boxplot: Min, 1st quartile, Median, 3rd quartile, and Max. Kruskal–Wallis tests. Source data are provided as a Source Data file. B Model . Stalled replication forks at PCS LT telomeres undergo SMARCAL1-dependent reversal, generating new 3′ telomeric ends. The reversed forks are bound by telomerase. Fork replication can resume through a homologous recombination-dependent restart process that requires RAD51 and might be facilitated by long 3′ overhangs generated by the telomerase and the formation of RNA:DNA hybrid formation, possibly involving TFIIH. If reversal is suppressed, PrimPol-dependent repriming can promote fork restart. This process may also be dependent on RNA:DNA hybrids. Optimal levels of TRF1 are required to suppress telomere fragility, by facilitating the reversal/restart processing of stalled forks. Protein illustrations were generated using BioRender (Vaurs, M. (2025) https://BioRender.com/ke04c93 ).

Article Snippet: One hundred microliters of lysate were kept as input and 900 μl were incubated with 1 μg of anti-TRF1 antibody (St John’s laboratory) for 4 h at 4 °C.

Techniques: Control, Homologous Recombination, Generated