Fig 2: EXD2 is recruited to dysfunctional telomeres and co-localises with ALT-associated PML bodies.a GFP-EXD2 (green) localises to ALT-associated PML bodies as determined by 4-colour immunofluorescence staining carried out in control U2OS cells expressing GFP only and U2OS cells stably expressing GFP-EXD2 using antibodies raised against TRF2 (red) and PML (magenta). DAPI (blue) acts as a nuclear stain (n = 3 independent experiments, scale bar = 10 μm). b Quantification of GFP/TRF2 co-localisations (percentage cells with co-localisations and average number of co-localisations per positive cell) in cells from (a), n = 150 cells examined over 3 independent experiments, bars represent +/− SEM. c Quantification of GFP/APB co-localisations (percentage cells with co-localisations and average number of co-localisations per positive cell; in cells from a), n = 150 cells examined over 3 independent experiments, bars represent +/− SEM. d, e GFP-EXD2 and FLAG-EXD2 localise to telomeres as assayed by its association with TRF2 (Shelterin component) by the PLA assay. The number of PLA foci per cell is quantified in U2OS control cells or cells stably expressing GFP- or FLAG-EXD2. PLA signal appears in red, DAPI (blue) acts as a nuclear stain (n = 194 vs. 204 cells (d) and 211 vs. 258 cells (e) examined over 3 independent experiments, statistical analysis was carried out by two-sided Mann–Whitney test, error bars represent +/− SEM, scale bar = 10 μm). Source data are provided as a source data file.

Fig 3: EXD2-deficient cells display increased telomere dysfunction.a Immuno-FISH staining in WT and EXD2−/− U2OS cells. 53BP1 (red) acts as a DSB marker, TelG PNA FISH probe staining (green) acts as a marker for the telomere and DAPI (blue) acts as a nuclear stain (n = 219, 172 and 175 cells examined over 3 independent experiments, statistical analysis by two-sided Mann–Whitney test, bars represent +/− SEM, scale bar = 10 μm). b PLA assay carried out in WT and EXD2−/− U2OS cells using antibodies recognising TRF2 (shelterin component) and 53BP1 (DSB marker). PLA signal appears in red, DAPI (blue) acts as a nuclear stain (n = 297, 259 and 281 cells examined over 3 independent experiments, statistical analysis was carried out by two-sided Mann–Whitney test, bars represent +/− SEM, scale bar = 10 μm). c Quantification of 53BP1/TelG co-localisations per nucleus by immuno-FISH staining in WT U2OS cells or cells stably overexpressing either WT or nuclease-dead mutant FLAG-HA EXD2, treated with either control siRNA or siRNA targeting the 3′UTR of endogenous EXD2 (n = 168, 152, 165, 190, 218, 223, 203, 181, 203 and 192 cells examined over 2 independent experiments. Statistical significance was determined by two-sided Mann–Whitney test, bars represent +/− SEM). d Quantification of sister telomere-free ends using telomere strand-specific PNA FISH probes (SpO and FITC-labelled, red, and green signals, respectively) in U2OS control and EXD2−/− cells (n = 75 metaphases per sample examined over 3 independent experiments, statistical analysis by two-sided Mann–Whitney test, bars represent +/− SEM, scale bar = 1 μm). e Quantification of RPA/TRF2 co-localisations (green and red signals, respectively) in U2OS WT and EXD2−/− cells. DAPI (blue) acts as a DNA stain (n = 303, 305 and 315 cells examined over 3 independent experiments, statistical analysis by two-sided Mann–Whitney test, bars represent +/− SEM, scale bar = 10 μm). f Immuno-FISH analysis of WT and EXD2−/− U2OS cells treated with DMSO or RAD51 inhibitor (B-02, 25 μM, 2 h treatment) or Mirin (50 μM, 1 h treatment). 53BP1 acts as a DSB marker, TelG PNA FISH probe staining acts as a marker for the telomere and DAPI acts as a nuclear stain (n = 301, 305, 300, 302, 302, 300, 300, 301 and 302 cells examined over 3 independent experiments, statistical analysis by two-sided Mann–Whitney test, bars represent +/− SEM). g Quantification of 53BP1 co-localisations with TelG telomeric probe by immuno-FISH staining in WT and EXD2−/− U2OS cells treated with either control siRNA or siRNA targeting SMARCAL1 (as indicated) (n = 150 cells examined over 3 independent experiments, statistical analysis was carried out by two-sided Mann–Whitney test). Source data are provided as a source data file.