(–)BI97D6 effectively induces apoptosis in AML cells regardless of high expression of Mcl-1. (A) Representative FACS plots showing gating strategy and apoptosis induction by (–)BI97D6 in OCI-AML3 cells. (B-C) Time- and dose-dependent (B) induction of apoptosis and (C) reduction of live cell numbers of OCI-AML3 cells after (–)BI97D6 treatment. (D) Mcl-1 expression in 3 AML cell lines as determined by immunoblot densitometry using the Odyssey Infrared Imaging System. α-Tubulin served as loading control. The ratio of Mcl-1/α-tubulin was normalized to that of untreated OCI-AML3 cells. (E-F) The effects of (E) ABT-737 and (F) (–)BI97D6 on 3 AML cell lines with low, intermediate, or high expression of Mcl-1 protein. Effect = 1 – live cell number^treated/live cell number^control. (G) Immunoblot showing stable Mcl-1 knockdown by lentiviral shRNA in OCI-AML3 cells. (H-J) Comparison of the sensitivities of OCI-AML3 shGFP and shMCL1 cells to ABT-737, (–)BI97D6, and Sabutoclax. The cells were treated with indicated concentrations for 72 hours. (K) Immunoblot showing stable Mcl-1 overexpression in HL-60 cells. (L-N) Comparison of the sensitivities of HL60-GFP and HL60-Mcl-1 overexpressing cells to ABT-737, (–)BI97D6, and Sabutoclax. The cells were treated for 48 hours. The live cell numbers were enumerated by FACS analysis using CountBright counting beads and normalized to those of untreated controls. Calcusyn software was used to calculate the IC₅₀ values based on live cell numbers. All data in line/bar graphs represent the means of triplicate experiments, with error bars indicating the standard deviations.