ATM kinase activation following Fas-induced apoptosis is a late passive event. (A) C3ABR cells were induced to undergo apoptosis with 250 ng/mL anti-Fas IgM monoclonal antibody. Untreated and NCS-treated cells that trigger DSB and classically induce ATM activation⁴²  were used as controls. For immunoblotting, 80 to 100 μg protein extract were separated by SDS-PAGE and transferred on nitrocellulose. The proteins of interest and their phosphorylation were revealed by immunoblotting with specific antibodies. (B) C3ABR cells were treated to undergo apoptosis with 250 ng/mL anti-Fas IgM monoclonal antibody (CH11; UBI). Untreated and treated cells were analyzed by flow cytometry for active caspase-3 and phospho-Ser1981-ATM. (C) C3ABR cells were treated to undergo apoptosis as in panel B. Untreated and NCS-treated cells were used as controls. Cells were fixed and permeabilized, and immunofluorescences were carried out as previously described.³²  Nuclear condensation and fragmentation have been evaluated by Hoechst staining.