TNF-α hypersensitivity of Fancc^-/- MEFs is Ask1 dependent. (A) Antioxidants and MEF apoptosis assays. WT and Fancc^-/- MEFs were grown in normal conditions, pretreated with 20 μM SeMet, or pretreated with 4 mM NAC before 50 ng/mL TNF-α treatment. Apoptosis was analyzed 24 hours after TNF-α treatment using TUNEL assay. For each experiment, at least 100 cells were evaluated (Hoechst⁺) to determine the percentage of apoptotic cells (TUNEL⁺) in each condition (n = 3; ^*P ≤ .002). (B) Ask1 in vitro kinase assays. Ask1 kinase activity was evaluated in WT and Fancc^-/- MEFs after treatment with 50 ng/mL TNF-α. Ask1 immunoprecipitations were subjected to an in vitro kinase reaction, as described. Autoradiography of Ask1 kinase assays, densitometry analyses, and Western blots for total Ask1 are shown. Data in the left panel are representative of 5 independent experiments. The graph on the right depicts the mean arbitrary density units from the 5 experiments (^*P < .001). (C) Dominant-negative Ask1 studies. WT and Fancc^-/- MEFs were transduced with a retrovirus encoding a catalytically inactive, dominant-negative Ask1 (Ask1-K709M) or vector control. Transduced MEFs were treated with 50 ng/mL TNF-α for 24 hours before evaluating apoptosis by the TUNEL assay (TMR-red⁺). The percentage of apoptotic cells was calculated by dividing EGFP⁺TMR-red⁺ cells by total EGFP⁺ cells for each condition. At least 100 cells were scored per condition per experiment (n = 3; ^*P < .01) compared with vector control + TNF-α. Data shown represent the mean of multiple experiments, and error bars represent the SD.