Modulation of ATM kinase activity results in the regulation of FLIP protein levels, which in turn determines Fas sensitivity. (A) Different cell lines were treated with NCS for 3 hours to trigger ATM kinase activation. Protein extract (80-100 μg) was separated by SDS-PAGE and transferred on nitrocellulose, and FLIP expression was revealed using specific antibodies. (B) C3ABR cells were stimulated to undergo apoptosis with 250 ng/mL anti-Fas mAb in the presence or in the absence of NCS pretreatment for 3 hours. Apoptosis was determined by the analysis of DNA fragmentation in PI-stained cells 24 hours after anti-Fas treatment. (C) The indicated cell lines were incubated in the presence of the specific ATM kinase inhibitor KU-55933 (10 μM) for 1 or 8 hours. Protein extract (80-100 μg) was separated by SDS-PAGE and transferred on nitrocellulose, and FLIP expression was revealed using specific antibodies. (D) C3ABR cells were preincubated for 1 or 8 hours with the specific ATM kinase inhibitor KU-55933 (10 μM) to allow endogenous ATM kinase inactivation and FLIP level up-regulation and then stimulated to undergo apoptosis with 250 ng/mL anti-Fas mAb. Apoptosis was determined by the analysis of DNA fragmentation in PI-stained cells 24 hours after anti-Fas treatment.