Fig. 2.

A, time course of γ-H2AX induction in vector and stable R2-knockdown p53(−/−) HCT-116 cells (vector and R2-kd, respectively, labeled in all figures and tables). Cells were pulsed with 30 μM cisplatin for 1 h and incubated until harvested at the indicated times. Total protein was analyzed by Western blotting to assess the levels of γ-H2AX, R2, and actin. B, the intensity of the γ-H2AX bands was quantified by densitometry and normalized against that of the actin bands. The normalized γ-H2AX induction plotted against time is shown. C, cisplatin-induced γ-H2AX occurred primarily in S and G₂/M phases of the cell cycle. R2-knockdown cells were exposed to 0 (red), 2.5 (blue), 7.5 (orange), and 15 (green) μM cisplatin for 24 h and 10 μM BrdU during the final hour. Cells were fixed, stained, and analyzed for bivariate distribution of BrdU incorporation and DNA content (top) by flow cytometry. G₁, S, and G₂/M cell populations were gated and further analyzed for the level of γ-H2AX in response to cisplatin treatment.