SPHK1 inhibition induces caspase-dependent cell death through MCL1 suppression in AML cell lines and primary human AML blasts. (A) AML cell lines MOLM-13, MV4-11, TF-1, UT-7, THP-1, and ME-1 were treated with increasing doses of MP-A08 for 48 hours, and caspase-3 activity was measured by substrate cleavage of NucViewTM488 (Biotium) and quantified by flow cytometry. Results are mean ± range. (B) MV4-11 cells were preincubated for 1 hour with the pan-caspase inhibitor Q-VD-OPh (25 μM) and subsequently treated with MP-A08 (30 μM) for 24 hours prior to analysis of cell survival by annexin V/PI staining. MV4-11 cells were incubated with increasing doses of MP-A08 (C) or SK1-I (D) for 16 hours following which whole cell lysates were immunoblotted with the indicated antibodies. (E) MV4-11 cells were incubated with MP-A08 (20 μM) for the indicated time following which whole cell lysates were immunoblotted with the indicated antibodies or MCL1 mRNA levels determined using quantitative RT-PCR (mean ± SEM, n = 3). (F) Primary AML blasts were incubated with 30 μM of MP-A08 for 16 hours following which whole cell lysates were immunoblotted with the indicated antibodies.