Proapoptotic effects of CI-1040 in combination with RAR- and RXR-selective ligands. (A) OCI-AML3 cells were pretreated with either vehicle (DMSO, □) or the MEK inhibitor CI-1040 (0.5 μM, ⊡) for 30 minutes and subsequently exposed to the following ligands for 96 hours: ATRA (1 μM), 9-cis RA (0.1 μM), TTNPB (1 μM), LG100268 (LG, 1 μM), methoprene acid (MA, 50 μM), 1,25 (OH)₂ vitamin D3 (vit D3, 0.1 μM), or CDDO (0.3 μM). Apoptosis was evaluated by flow cytometric analysis of FITC-conjugated annexin V binding, while simultaneously assessing membrane integrity by PI exclusion. Results are expressed as the percentage of annexin V–positive cells and represent the average ± SD of at least 3 independent experiments. Synergism analysis for (B) viability reduction and (C) apoptosis induction was carried out using a fixed dose of CI-1040 (0.5 μM) and escalating doses of TTNPB (0.1-1 μM, □) and LG100268 (0.1-1 μM, ▴); CI values were then derived using the Chou-Talalay method⁴⁴  and are plotted against ligand concentrations. The gray area indicates additive effects (CI = 0.9-1.2), while synergism and antagonism fall below and above the area, respectively.