U87MG cells (panels A to F) were reconstituted the indicated proteins. Cells treated with IR (5 Gy) were immunostained at the indicated times with antibodies to γH2AX, 53BP1, BRCA1 or RAD51. Cells containing >5 foci were scored as positive. Bars represent SEM. PTEN SUMOylation is required for the resolution of 53BP1 foci (p=0.0014, t test, n=3) (A), γH2AX foci (p=0.007, t test, n=3) (B) and BRCA1 foci (p=0.0136, t test, n=3) (C). (D) PTEN SUMOylation is required for RAD51 focus formation. (p=0.0016, t test, n=3). (E) Impaired homologous recombination-based DNA repair in PTEN- and SUMO-PTEN-deficient cells. U87MG cells stably expressing a DR-GFP reporter were reconstituted with the indicated PTEN proteins and their HR-mediated repair assessed as previously described (16). HR efficiency is expressed relative to that of wt. (p=0.012, t test, n=2). (F) Lack of SUMO-PTEN increases radiosensitivity. Surviving fraction was determined by Sulforhodamine B staining 6 days following exposure to 3 Gy of IR (p=0.02, t test, n=3).