Fig. 4

Evaluations of apoptotic response induced by simultaneous inhibition of AKT/mTOR and WNT/β-catenin pathways. a MOLM-14 cells were treated either with DMSO control, VS-5584, ICG-001 single agent or in combination for 48 h, harvested for staining with annexin V/FITC and PI dye as described in the “Methods” section. Upper representative images show the original FACS plots and lower bar figures represent the percentage of annexin V-positive cells, including both early and late apoptotic cells. b Luminescent assays for caspase 3 and caspase 7 activities in MOLM-14 cells and primary AML cells from unique patient number 4 (Pt#4, PRL-3 high) (c) incubated with either VS-5584, ICG-001 alone, or combination (n = 3, mean ± SD). Five-point constant ratio of combination-based single drug IC₅₀ were used here. V + I: combination of VS-5584 and ICG-001. d Caspase 3 and caspase 7 activities in primary AML cells from unique patient number 9 (Pt#9, PRL-3 low) incubated with either VS-5584, ICG-001 alone, or combination (n = 3, mean ± SD). Five-point constant ratio of combination-based single drug IC₅₀ were used here. V + I: combination of VS-5584 and ICG-001. e The cell lysates extracted from MOLM-14 cells treated with DMSO control, VS-5584 (800 nM), ICG-001 (14 μM) single agent or in combination for 48 h were subjected to Western blot analysis for AKT, p-AKT, Survivin, full length (FL) or cleaved (CL) caspase 3, 7 and PARP. Beta-actin was used as the loading control. Protein levels were determined by densitometric analysis. The experiments were duplicated, and representative images were shown