Bid is cleaved in a caspase-9-dependent fashion during IL-3 withdrawal and is necessary for ROS production. (A-B) FL5.12 Neo (±100 μM BocD-fmk), Bcl-xL, Casp9DN and CrmA cells were cultured in the presence or absence of IL-3 for 24 h. (A) Bid levels were determined by western blot and viability was determined. (B) Loss of membrane potential was determined by flow cytometry (filled histogram (+)IL-3, black line (-)IL-3, grey line (-)IL-3 +BocD). (C)In vitro translated Bid, BidD98A, BidD75A and BidD59A were subjected to cleavage by increasing levels of caspase-9 for 90 min. Bid cleavage was determined by western blot. (D) Bid and BidD59A were expressed in Bid-/- MEFs by retroviral transduction and Bid levels were determined by western blot (Bid expression from same blot). (E) Bid-/- pBabe, Bid and BidD59A MEFs were withdrawn from serum for 12 hr. ROS production was determined by flow cytometry. (light grey line (+)FBS, dark grey line (-)FBS, black line (-)FBS +BocD). Treatment with 50 μg/ml antimycin A for 30 min was used as a positive control (red line). Data are representative of at least 3 independent experiments.
Brentnall et al. BMC Cell Biology 2013 14:32 doi:10.1186/1471-2121-14-32