Fig. 3.

Cells lacking Brca1 enzymatic activity are resistant to MMC-induced genotoxic stress. Isogenic ES cells that are proficient (Brca1^FH-WT/−) or deficient (Brca1^FH-I26A/−) for Brca1 E3 ligase activity were examined for MMC sensitivity in clonogenic survival assays, together with ES cells homozygous for a hypomorphic Brca1 mutation (Brca1^{Δ223–763/Δ223–763}) and control ES cells (Brca1^+/−). Cells were plated and treated with various concentration of MMC for 4 h. After 8–10 days, the surviving colonies were stained with Giemsa and counted. Survival is calculated as a percentage of colonies in the mock-treated plates. Each subclone was tested in triplicate, and the error bars represent SE of the mean of survival for each subclone. Similar results were also observed in separate experiments by using additional independently derived Brca1^FH-WT/− and Brca1^FH-I26A/− ES subclones.