Ectopic expression of dominant-negative Ras (S17N) prevents ERK1/2 activation and enhances γH2A.X expression following exposure to Chk1 inhibitors. (A,B) U266 cells were stably transfected with S17N H-Ras or its empty vector (EV), and ectopic expression of mutant protein was detected by WB (A, top panels). Cells were then exposed to UCN-01 or Chk1i for 24 hours, after which Ras activation assay and WB were performed to monitor Ras activity (A, bottom panel) and expression of phospho-ERK1/2 and γH2A.X, as well as PARP cleavage. The results of representative experiments are shown; 2 additional studies yielded equivalent results. CF indicates cleavage fragment. (C,D) Cells were incubated with UCN-01 or Chk1i for 24 hours and 48 hours, after which percentage of annexin V–FITC–positive cells was determined by flow cytometry. The results represent the means plus or minus SD for 3 separate experiments performed in triplicate. *P < .05; **P < .01.