Fig. 4: EXD2-deficiency switches pathway choice from canonical HR towards BIR at telomeres in ALT-positive cells. | Nature Communications

Fig. 4: EXD2-deficiency switches pathway choice from canonical HR towards BIR at telomeres in ALT-positive cells.

From: Pathway choice in the alternative telomere lengthening in neoplasia is dictated by replication fork processing mediated by EXD2’s nuclease activity

Fig. 4

a Immuno-FISH in WT and EXD2−/− U2OS cells synchronised to G2 by RO-3306 treatment and labelled with EdU as indicated. EdU incorporation was determined by Click-iT staining (red) and telomeres marked by a telomere-specific PNA FISH probe (TelG, green) with co-localisation indicated by arrows. Cells were treated with control siRNA or siRNA targeting RAD52 as indicated. DAPI (blue) acts as a DNA stain (n = 164, 155, 120, 130, 108 and 99 cells examined over 3 independent experiments, statistical significance was determined by two-sided Mann–Whitney test, bars represent +/− SEM, scale bar = 10 μm). b Immuno-FISH analysis in WT or EXD2−/− U2OS cells synchronised to G2 by RO-3306 followed by release to synchronised prometaphase in the presence of EdU as indicated. EdU incorporation was determined by Click-iT staining (red) and telomeres marked by a telomere-specific PNA FISH probe (TelG, green) with co-localisations indicated by arrows. DAPI (blue) acts as a DNA stain. Cells were treated with DMSO, or RAD52-inhibitor (n = 140, 157, 144, 118, 86 and 83 prometaphases examined over 3 independent experiments, statistical significance was determined by two-sided Mann–Whitney analysis, bars represent +/− SEM, scale bar = 10 μm). c Quantification of conservative telomeric synthesis in WT vs EXD2−/− U2OS cells and RAD52−/− Cyclin E-overexpressing U2OS vs parental control cells as determined by two-replication round segregated CO-FISH using SpO and FITC-labelled PNA FISH probes (red and green signals, respectively) specific to the telomeric repeat sequences (n = 75 metaphases examined over 3 independent experiments, statistical analysis was carried out by two-sided Mann–Whitney analysis, bars represent +/− SEM, scale bar = 10 μm). Source data are provided as a source data file.

Back to article page