Figure 3
DTX induces lysosomal membrane permeabilization (LMP) in PC3 cells. A. Determination of lysosomal integrity in PC3 cells treated with DTX for 24 hours in the presence and absence of Z-VAD-FMK. Cells were exposed to acridine orange (AO) for lysosome visualization under fluorescence microscopy. Untreated cells showed localized granular red fluorescence corresponding to staining of intact lysosomes. Cells that detached after treatment with DTX in the presence and absence of Z-VAD-FMK displayed increased yellow fluorescence indicative of LMP. Most cells that remained attached after treatment appeared to have intact lysosomes but exhibited multinucleation. Hoescht staining was used to visualize nuclear morphology: B. Cathepsin B inhibitor (CBI) but not inhibitors of cathepsin L (CLI) or cathepsin D (CDI) attenuated cell death induced by 0.1 μM DTX (top set of graphs) and 3 μM DTX (bottom set of graphs), as assessed by crystal violet viability assays. Errors bars represent the standard deviation of at least three independent experiments done in triplicate (*p < 0.05, t-test): C. Detection of intracellular cathepsin B activity in PC3 cells using the fluorogenic substrate Magic Red MR-(RR)2. Cells treated with DTX for 36 hours showed diffuse cathepsin B activity (red fluorescence), whereas the activity in untreated cells and cells treated with DTX in the presence of CBI was localized mainly to lysosomes.