Inhibition of NPM/ALK signaling promotes Bcl-XL/Bad heterodimerization and disruption of mitochondrial homeostasis. (A) K-299 cells were incubated with vehicle alone (DMSO), herbimycin A (10 μM), LY294002 (25 μM), or left untreated (NT) for 8 hours. Mitochondrial extracts (left panels) and total lysates (right panels) were immunoprecipitated by using an anti-Bcl-XL polyclonal antibody. Supernatant (IP:sup) depleted of Bcl-XL complexes and immunoprecipitates (IP: Bcl-XL) were analyzed by Western blot with anti-Bcl-XL and Bad antibodies. (B) Disruption of mitochondrial transmembrane potential (ΔΦm) was assessed after an 8-hour incubation with LY294002 (25 μM) or the equivalent amount of DMSO by FACS analysis of JC-1 dye-stained cells. Apoptotic cells fall into the R5 region (right panel) showing a reduced red FL2-H-fluorescence: R5 = 2.1%, in the control sample (upper right panel) and 47.6% in the LY294002-treated sample (lower right panel). The left panels show cells plotted as forward scatter (FSC) against side scatter (SSC) showing no substantial alteration in cell morphology at this early stage of apoptosis induction.
