Specific antisense oligonucleotide-mediated down-regulation of Bcl-X_L increased apoptosis of NPM/ALK⁺ cells in vitro. (A) Ba/F3-NPM/ALK⁺ cells were incubated for 72 hours with increasing concentrations of either sense (S), nonsense scrambled (NS), or antisense (AS) phosphorothioated oligonucleotides (ODNs) targeting the Bcl-X_L AUG region. Fresh oligonucleotides were added at 0, 16, 24, 32, 48, and 60 hours. Western blot analysis was performed 72 hours after treatment. Blots were reprobed with an anti-β-actin specific antibody as control for protein loading. (B) Total cell lysates from Ba/F3-NPM/ALK⁺ cells treated with 30 μM Bcl-X_L AS-ODN for 48, 52, and 60 hours were compared with those of cultures incubated with sense (S) and nonsense scrambled (NS) ODNs for 60 hours for Bcl-X_L expression by immunoblotting. (C) The fraction of apoptotic cells was evaluated by annexin V-propidium iodide staining and flow cytometric analysis after 96 hours of treatment with ODNs or in the untreated control (NT). The values indicate the percentages of cells in early (lower right quadrant) and late (upper right quadrant) apoptosis. (D) Ba/F3-N/A⁺ cells treated for 48 hours with Bcl-X_L antisense, control nonsense scrambled, or no oligodeoxynucleotides (oligo) were incubated with increasing concentration of etoposide for additional 18 hours. Apoptosis was evaluated by AO staining at the end of drug incubation; the data are represented as the mean ± standard deviation (SD) of 3 independent experiments.