Figure 3. ART-838 induced Caspase-mediated apoptosis and ROS generation in MOLM14 cells more potently than did AS.

A-B. Cells were pretreated with z-VAD (50 μM) for 1h, and then incubated with ART-838 (0.1 μM) or AS (5 μM) for 24h. A. Quantification of the percentage of early apoptotic cells (Annexin-V+/7-AAD-) from three independent experiments in which cells were stained for apoptosis with 7-AAD and Annexin-V then analyzed by flow cytometry. (B) Cells were analyzed for Caspase-3/7 activity using a fluorescent peptide substrate after z-VAD pretreatment and 24h ART-838 or AS treatment. C. Cells were pre-loaded with CM-H₂DCFDA (5 mM) for 30min, pretreated with DFO (11 mM) or NAC (25 μM) for 60-120min, and then treated with ART-838 (IC₉₀: 0.1 μM) or AS (IC₉₀: 5 μM). Preliminary experiments showed that DFO and NAC were cytotoxic to MOLM14 cells at high concentrations, so each was used at its approximate 48h IC₅₀ (concentration that inhibited growth by only 50% at 48h). 16h after addition of drugs, cells were washed and analyzed by flow cytometry for CM-H₂DCFDA fluorescence (indicating total ROS generation) in the FL1 channel. Mean Fluorescence Intensity (MFI) was normalized to that of vehicle control (0.5% DMSO) with no pretreatment. D. Cells were pretreated with DFO or NAC as in C, then treated with ART-838 (IC₅₀: 0.025 μM, IC₉₀: 0.1 μM) or AS (IC₅₀: 0.825 μM, IC₉₀: 5 μM) for 24h before analyzing for apoptosis as in A. E, F. Cells were pretreated with DFO or NAC as in C, then incubated with a range of concentrations of AS (E) or ART-838 (F). for 48h. Viable cell counts, obtained via Trypan Blue dye exclusion, were normalized to vehicle (0.02% DMSO)-treated samples +/– pretreatment. [**, p<.01 and ***, p<.001 for ART-838 or AS vs control (far left bars); #, p<.05, ##, p<.01, and ###, p<.001 for ART-838 or AS with vs without inhibitor pretreatment].