Flow cytometric assays showing induction of death, apoptosis, and cell cycle changes by single-agent obatoclax in MLL-AF4 ALL cell lines. (A) Surviving fraction plots of cell viability measured by MTT assays at 72 hours after obatoclax or ADR (positive control for apoptosis) exposure. Assays were performed at least 3 times (6 replicates/condition per experiment). Error bars indicate standard error. Results from MTT assays at 72 hours determined the low and high obatoclax (EC₅₀; 3 × EC₅₀) concentrations and the ADR concentration that were used in time-course flow cytometric assays (B-E). (B) Flow cytometric assays of cell death measured by PI fluorescence uptake in vehicle-, obatoclax-, or ADR-treated cell lines. Average percentage of dead (PI positive) cells from 5 independent experiments are plotted. (C) Flow cytometric assays of activated caspase 3 in vehicle-, obatoclax-, or ADR-treated cell lines. Activated caspase 3 positive cells were gated to determine percentages of apoptotic cells. Graphs represent average percentage of caspase 3 positive cells from 5 independent experiments. (D) FACS TUNEL assay of DNA fragmentation in vehicle-, obatoclax-, or ADR-treated cell lines. TUNEL-positive cells were gated to determine percentages. The average percentage of TUNEL positive cells from 4 independent experiments is plotted. Bars (B-D) show standard error. Asterisk indicates P ≤ .05 vs vehicle at individual points. (E) Representative cell cycle analyses in vehicle-, obatoclax-, or ADR-treated cell lines with cell cycle distribution determined using ModFit LT. (F) Bar graph plots of percentages of cells in G0/G1, S, and G2/M in vehicle-, obatoclax-, or ADR-treated SEM-K2 cells in 6 independent experiments.