Fig 1: TRF2 phosphorylation at S410 increases its binding to TIN2. (A) Biotin-Ahx-ISRLVLEEDpSQSTEPSAGLN (TRF2-pS410) and Biotin-Ahx–SRLVLEEDSQSTEPSAGLN (TRF-CTRL) peptides were incubated with nuclear extracts and pulled down by streptavidin beads. Bound proteins were identified by mass spectrometry (n = 3). Plotted are –log P values of proteins enriched or reduced in condition with TRF2-pS410 peptide. The line delineates the statistical significance (FDR < 0.1). (B) Fluorescently-labelled TRF2-pS410 and TRF2-CTRL peptides were titrated with purified TIN2 to a final concentration of 500 nM. Fluorescence anisotropy change was measured and dissociation constant values for unmodified and modified oligopeptides were calculated as described in Methods. (C) HEK293 cells stably expressing EGFP-TRF2 were treated with DMSO or with PPM1D inhibitor for 4 h. EGFP-TRF2 was immunoprecipitated from cell extracts with GFP Trap. Proteins were separated by SDS-PAGE and binding of TIN2 was determined by immunoblotting. Numbers at the bottom indicate the TIN2 signal relative to the total immunoprecipitated TRF2 and normalized to the wild-type TRF2. Representative result from three experiments is shown. (D) TRF2:TIN2 interaction was determined in parental U2OS and U2OS-PPM1D-KO cells by PLA. Mean PLA foci count is plotted ± SD, n = 500. Statistical significance was evaluated using Mann–Whitney test (****P < 0.0001). Representative experiment is shown from two independent repeats. (E) TRF2:TIN2 interaction was determined in U2OS cells treated with DMSO or PPM1Di by PLA. Mean PLA foci count is plotted ± SD, n = 500. Statistical significance was evaluated using Mann–Whitney test (****P < 0.0001). Representative experiment is shown from two independent repeats. (F) U2OS cells were treated or not with PPM1Di for 24 h, pre-extracted, fixed and stained with TRF2 (m-Santa Cruz) and TIN2 (Rb-Novus) antibodies. Mean TIN2 intensity in TRF2 foci is plotted ± SD, n = 300. Statistical significance was evaluated using Mann–Whitney test (****P < 0.0001). Representative experiment is shown from two independent repeats. The scale bar represents 10 µm. (G) Parental U2OS, U2OS-PPM1D-KO and U2OS-PPM1D-KO stably expressing FLAG-PPM1D cells were treated or not with PPM1Di for 24 h. Cells were pre-extracted, fixed and stained for TIN2 and TRF2. Mean TIN2 intensity in TRF2 foci ± SD is plotted, n = 300. Statistical significance was evaluated using Mann–Whitney test (****P < 0.0001). Representative experiment is shown from two independent repeats. (H) Levels of TRF2 and TIN2 were analyzed in whole cell extracts from the parental U2OS and U2OS-PPM1D-KO cells by immunoblotting. Importin staining was used as a loading control. (I) Cells from G were analysed for TRF2 intensity in TRF2 foci. Plotted is mean ± SD, n = 300. (J) U2OS cells were treated or not with PPM1Di for 24 h, pre-extracted, fixed and stained with TRF2 and TPP1 antibodies. Mean TPP1 intensity in TRF2 foci normalized to the mean nuclear TPP1 intensity ± SD is plotted, n > 300. Statistical significance was evaluated using Mann–Whitney test (****P < 0.0001). The scale bars in representative images corresponds to 10 µm and 1 µm respectively. (K) Parental U2OS, U2OS-PPM1D-KO cells and U2OS-PPM1D-KO stably expressing FLAG-PPM1D cells were pre-extracted, fixed and stained for TPP1 and TRF2. Mean TPP1 intensity in TRF2 foci normalized to the mean nuclear TPP1 intensity ± SD is plotted, n > 300. Statistical significance was evaluated using Mann–Whitney test (****P < 0.0001, ***P < 0.001). (L) Chromosome spreads from parental U2OS and U2OS-PPM1D-KO cells were hybridized with TAACCC FISH-probe and imaged by 3D-SIM. Plotted is a fraction of telomeres that formed t-loops. More than 203 telomeres were quantified per condition in each experiment (n = 3). Significance was determined by unpaired t-test.