Figure 7. ATR inhibition synergizes with H-ras^G12V, K-ras^G12D, and c-Myc overexpression to cause increased genomic instability and cell synthetic lethality.

(A) Phosphorylation of H2AX in response to ATR inhibition in the context of oncogene expression. NIH3T3 cells were transduced with retroviruses expressing the indicated oncogenes or empty control vector (pBabe-puro). Following drug resistance marker selection, cell lines were expanded and treated with 1 μM ATR-45 inhibitor for 7 hours. Cells were then harvested for Western blot detection of the indicated proteins. (B) Cell-cycle distribution of γH2AX following ATR inhibition. Oncogene-expressing and control cell lines were treated with 4 μM ATR-45 inhibitor for 24 hours and detected for phospho-S139 H2AX and DNA content (propidium iodide staining). Aphidicolin (0.5 μM) was added to control cells to induce exogenous replication stress and serve as a positive control for its effects. (C) Chromatid breaks following short-term ATR inhibition in oncogene-expressing and control cell lines. Cell lines were treated with 2 μM ATR-45 inhibitor for 7 hours, as described in A, were harvested for mitotic spreads, and chromatid breaks were quantified. Nocodazole (0.5 μM) was added 4 hours prior to harvest. Data represent mean ± SEM.