FIGURE 3.

WIP1 reverses ATR-mediated γ-H2AX phosphorylation. A and B, WIP1 suppresses γ-H2AX phosphorylation after UV (A) and hydroxyurea treatment (B). HeLa cells were transfected with empty vector or WIP1-FLAG expression plasmid. At 24 h after transfection, cells were irradiated with 50 J/m² of UV (A) or treated with 10 mm hydroxyurea (HU) (B) and harvested at the indicated time points. Nuclear lysates were immunoblotted as indicated. C, WIP1 suppresses γ-H2AX phosphorylation in the absence of ATM. GM09607 (ATM null) fibroblasts were transfected with either empty vector or WIP1-FLAG expression plasmid. After 24 h, cells were irradiated with 50 J/m² of UV and harvested. Nuclear lysates were immunoblotted as indicated. D, suppression of WIP1 in MCF-7 cells results in enhanced γ-H2AX phosphorylation following UV irradiation. MCF-7 cells were transduced with one of two different shRNA lentiviral vectors prior to 10 J/m² UV irradiation. Nuclear lysates were harvested 1 h post-UV treatment along with un-irradiated control cells and mock transduced controls. Immunoblot analysis for WIP1 protein levels, H2AX protein levels, and γ-H2AX levels was performed.