Figure 6

CPT induces mitochondrial membrane potential and induces caspase-9 activation. Tetramethylrhodamine, ethyl ester, perchlorate (TMRE) was used to determine mitochondrial membrane potential (∆Ψm). A, Mitochondrial membrane potential depolarization in autophagy-competent and autophagy-inhibited DLM8 cells following 24 h CPT treatment. Following 24 h CPT treatment, cells were incubated with TMRE followed by flow cytometry analysis. Decreased TMRE fluorescence is indicative of decreased ∆Ψm and increased release of pro-apoptotic molecules into the cytosol. Cells were incubated with the membrane uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) prior to TMRE incubation to depolarize the mitochondrial membrane and serve as a positive control. Open histogram represents CCCP + TMRE treatment. Filled histogram represents cells incubated with TMRE only. Percentage values represent degree of mitochondrial membrane depolarization. Data is representative of results from two independent experiments. B, Caspase-9 activation and caspase-3 activation is reduced in autophagy-inhibited DLM8 cells. C, Caspase-3 activation is increased in autophagy-inhibited K7M3 cells. Cells were treated with CPT for 48 h. Cells were next collected, lysed and total protein immunoblotted for cleaved caspase-9 or cleaved caspase-3. Actin served as a protein loading control. Immunoblots are representative of immunoblots from at least two independent experiments.