BM MSC coculture increases Mcl-1 protein level in OCI-AML3 cells and Mcl-1 inhibition by the pan–Bcl-2 inhibitor (–)BI97D6 abrogates BM MSC-mediated protection. (A) Representative FACS plots showing the gating strategy used to determine apoptosis and viability of OCI-AML3 cells cocultured with BM MSCs. (B) Viability of OCI-AML3 cells following treatment with increasing concentrations of (–)BI97D6 for 72 hours, with or without coculture with protective MSCs. The live cell number (AnnV⁻/PI⁻) was enumerated by FACS analysis using CountBright counting beads and normalized to the counts of untreated controls. (C) Apoptosis (AnnV⁺) as assessed by FACS in OCI-AML3 cells after incubation with indicated compounds for 72 hours; cells were cultured alone or cocultured with protective MSCs. (D) Live cell numbers after incubation with indicated compounds for 72 hours. The methodology is as described in B. Both Sabutoclax and (–)BI97D6 effectively killed OCI-AML3 cells cocultured with BM MSCs. (E) Coculture with BM MSCs for 24 hours significantly increased Mcl-1 protein level in OCI-AML3 cells. (F-H) (–)BI97D6 (100 nM) had minimal effects on BM MSC (F) morphology, (G) proliferation, and (H) viability. NS, not significant. All data in bar graphs represent the means of triplicate experiments, with error bars indicating the standard deviations.