Figure 5. Rad17 promotes ATM activation and DNA end resection.

A, B Rad17 is required for activation of ATM signaling. U2OS cells transfected with the indicated siRNA for 2 days were either untreated or exposed to 5 Gy of IR. Western blot was performed as indicated (A). Immunofluorescence staining was performed, and the percentage of p-ATM foci-positive cells was plotted (mean ± s.d., n = 3) (B).

C U2OS cells transfected with indicated siRNAs were either untreated or exposed to 5 Gy of IR. Cell lysates were prepared at indicated times, and Western blotting was performed.

D–F Rad17 depletion significantly altered DNA end resection after IR. U2OS cells transfected with indicated siRNA were either untreated or exposed to 5 Gy of IR. Western blot was performed with anti-RPA pS4/8 as indicated (D). Immunostaining was performed as indicated, and the percentage of RPA foci-positive cells was plotted (mean ± s.d., n = 3) (E). Cells were grown in culture medium containing 10 μM BrdU for 24 h before IR (5 Gy) exposure. After IR, immunostaining was performed with anti-BrdU antibody as indicated according to the method as described (Hu et al, 2011) (F).

G U2OS cells were transfected with indicated siRNAs for 2 days. HR repair assay was performed as previously described.

H U2OS cells transfected with control siRNA or ATR siRNA were either left untreated or exposed to 5 Gy of IR. Western blot was performed as indicated.

Source data are available online for this figure.