ELL-12 triggers cytosolic accumulation of cyt c and caspase-9 activation that are inhibited by Bcl-xLoverexpression. Jurkat T cells treated with 10 μg/mL ELL-12 were harvested at the indicated times, and cytosolic and mitochondrial proteins were separated by 15% SDS-PAGE and analyzed by immunoblotting with anti-cyt c (A) or anti-cyt c oxidase (subunit II; B). Cyt c oxidase serves as a marker for mitochondrial contamination of cytosolic extracts. A mitochondrial extract from nontreated cells (mit. fr. Con) was used as a positive control for cytc and cyt c-oxidase (subunit II). Jurkat/neo and Jurkat/Bcl-xL cells, treated without or with 10 μg/mL ELL-12 for 2 hours, were harvested and cytosolic proteins were separated by 15% SDS-PAGE and analyzed by immunoblotting with anti-cytc (C). Cytosolic extracts from Jurkat/neo or Jurkat/Bcl-xL cells treated for 5 hours without or with 10 μg/mL of ELL-12 were subjected to SDS-PAGE and immunoblotted with anti–caspase-9 (D). Cytosolic and mitochondrial proteins from H9 cells treated with 10 and 50 μg/mL ELL-12 for 16 hours were separated by 15% SDS-PAGE and analyzed by immunoblotting with anti-cyt c(E) or anti-cyt c oxidase (subunit II; F). Cytosolic extracts from H9 cells treated for 16 hours without or with 10 and 50 μg/mL of ELL-12 were subjected to SDS-PAGE and immunoblotted with anti–caspase-9 (G). The migration positions of full-length caspase-9, cleavage intermediate p35, and active subunit p10 are indicated.
