Inhibition of Rac1 by N17Rac1 mutant or Rac1 siRNA diminishes IR-induced G
/M checkpoint activation. (A) MCF-7 cells were infected with Ad.N17Rac1 or Ad.Control for 24 hours and exposed to 15-Gy IR. Left panel: the cells were analyzed for DNA content 24 hours after IR. The result depicts the percentage of cells with 4N-DNA content and is shown as mean ± SD of quadruplicate samples. * P < 0.001 (n = 4), significant difference from the irradiated Ad.Control-infected cells. Right panel: Inset: at 15 minutes after IR, the infected cells were analyzed for Rac1 activities (Rac1-GTP) and protein levels (total Rac1). Bar graph: mitotic cells in the cell samples were analyzed 2 hours after IR. The result depicts the percentage of mitotic cells and is shown as mean ± SD of triplicate samples. ** P = 0.002 (n = 3), significant difference from the irradiated Ad.Control-infected cells. (B) Upper panel: MCF-7 cells transfected with Rac1 siRNA (Rac1) or control siRNA (Control) were incubated for the indicated times and analyzed for protein levels of Rac1 and Actin. Lower panel: After 2-day incubation, the siRNA-transfected cells were exposed to IR, incubated for 24 hours, and assessed for DNA content. Results depict the percentage of cells with 4N-DNA content and represent the mean ± SD of three separate experiments in duplicate samples. * P < 0.001 (n = 6), significant difference from the irradiated Control-siRNA transfected cells. (C) After 2-day incubation, siRNA-transfected cells were treated with/without 20-Gy IR, incubated for 1 hour, and analyzed for ATM, ATR, Chk1, and Chk2 activities.