FIGURE 7.

PTIP is important for proper DNA damage response after ionizing radiation. A, PTIP and PA1 play a minor role in G₂/M checkpoint control after DNA damage. PTIP^+/+, or PTIP^-/-, and ATM^-/- MEFs were mock-treated or irradiated (3 Gy). Similarly, U2OS cells transfected with control siRNA (siCTR), PTIP specific siRNAs (siPTIP#1 and siPTIP#2), or PA1-specific siRNAs (siPA1#1 and siPA1#2) were also mock-treated or irradiated. Cells were harvested 1 h later, and a G₂/M checkpoint assay was carried out (see “Materials and Methods”). The percentages of cells stained with phospho-H3 antibody before and after IR treatment were obtained from three individual experiments. Error bars indicate S.D. B, PTIP and PA1 are required for cell survival after ionizing radiation. PTIP^-/- cells were reconstituted with wild-type or various PTIP deletion mutants. Cell survival after irradiation was measured by clonogenic assay according to the “Materials and Methods.” The expression of PTIP or its deletion mutants in these cells were confirmed by Western blotting using indicated antibodies (lower panel). Similarly, U2OS cells were treated with control or PA1 specific siRNAs. Cell survival after IR was measured by clonogenic assay (upper panel), and Western blot analysis was performed to verify the depletion of PA1 after siRNA transfection (right panel). WT, wild type. C, a proposed model of the DNA damage responsive pathway involving PTIP. See “Discussion” for details.