(–)BI97D6 induces multiple hallmarks of mitochondrial apoptosis. (A-B) Phosphatidylserine externalization as determined by (A) Annexin V (AnnV) staining and (B) loss of mitochondrial membrane potential as measured by CMXRos assay in OCI-AML3 cells after 48-hour treatment with 50 nM (–)BI97D6. (C) Cleavage of caspase-3, caspase-9, and PARP-1 in OCI-AML3 cells after (–)BI97D6 treatment of 36 or 48 hours. (D) Apoptosis induction (AnnV⁺) by 50 nM (–)BI97D6 in OCI-AML3 cells pretreated with a caspase-3 or -9 inhibitor for 2 hours. ***P < .001. (E) Bax conformational change as measured by immunoprecipitation (IP) with an anti-Bax 6A7 monoclonal antibody. OCI-AML3 cells were treated with 100 nM (–)BI97D6 for 24 hours. WCL, whole cell lysate. (F) Disruption of Bcl-2/Bax association by (–)BI97D6 as shown by Bax/Bcl-2 co-IP. Bax was immunoprecipitated from OCI-AML3 cells following (–)BI97D6 treatment of 24 hours. (G) Disruption of Mcl-1/Bim association by (–)BI97D6. Bim was immunoprecipitated from OCI-AML3 cells treated with 100 nM (–)BI97D6 or vehicle for 24 hours. (H-I) Bim deficiency reduced apoptosis induction by (–)BI97D6. (H) Representative flow cytometry plots of MEF control and Bim^−/− cells following treatment with 300 nM (–)BI97D6 for 48 hours. (I) Apoptosis induction as measured by AnnV/PI flow cytometry after 48-hour treatment with 300 nM (–)BI97D6.