FIG. 6.

Response to γ-irradiation and MNNG in Brca1^S971A/S971A MEFs and tumor cells. (A to D) Partially defective G₂/M checkpoint activation in Brca1^S971A/S971A MEFs. (A) Mitotic index of irradiated or unirradiated wild-type (WT) and Brca1^S971A/S971A MEFs was determined at the indicated times after 10 Gy of IR. The mitotic index of irradiated (B) or unirradiated (C) wild-type and Brca1^S971A/S971A MEFs was determined in the presence of nocodazole (100 ng/ml). Means ± standard errors of the mean from at least three different littermate experiments are given. Significant differences are indicated by asterisks. (D) Representative fluorescence-activated cell sorter plots of wild-type and Brca1^S971A/S971A MEFs 4 h after 0 or 10 Gy of irradiation. For each analysis, 10,000 cells of each genotype were analyzed. (E) DNA synthesis was measured 1 h after the indicated dose of irradiation in wild-type and Brca1^S971A/S971A MEFs. (F) Expression patterns of p53 and p21 after treatment with γ-irradiation (10 Gy). C and IR, unirradiated and irradiated MEFs, respectively. (G) p53 expression levels in wild-type (W) and Brca1^S971A/S971A (M) cells in the absence (CTL) and presence (MNNG) of MNNG for 12 h were measured and compared as described in Materials and Methods. (H) p53 levels in wild-type and Brca1^S971A/S971A MEFs from 0 to 12 h after MNNG (250 μM) treatment. (I) Levels of p53 after treatment with MNNG in wild-type and Chk2^−/− MEFs generated independently from two different littermates.