Figure 4

Analysis of the apoptotic pathway in LBH589-induced cell death. Cells were treated with either vehicle or the indicated concentrations of LBH589 for 24–48 h. After cells were harvested, flow cytometric analysis (FCM) (a, d, e and f) or western blotting (b and g) was performed. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (100 ng/ml)-treated Jurkat cells were used as a positive control of typical apoptosis and LBH589 (12 nM)-treated K562 cells were used as a negative control (c, d and f). (a) Changes in expression of Fas were indicated using RFI (the ratio of mean fluorescence intensity for specific staining to that for control staining). (d) Activities of caspases. Cells were incubated with the IETD-FMK for caspase-8, conjugated to FITC (FITC-IETD-FMK) or the LEHD-FMK for caspase-9, conjugated to FITC (FITC-LEHD-FMK) and analyzed using FCM. Comparison of the fluorescence intensity in the treated sample with that of the untreated control allows determination of the fold increase in activity of each caspase. (e and f) Mitochondrial membrane permeability. Cells were incubated with the JC-1 dye and analyzed using FCM. The percentage of cells with low JC-1 red fluorescence was evaluated. (b, c and g) Western blotting was performed using whole-cell lysate (b and c) or a cytosolic fraction (g) prepared as described in Materials and methods.