Figure 3

Camptothecin increases autophagic activity. Following 48 h CPT treatment, cells were incubated with the lysosomotropic agent acridine orange and fluorescence analyzed by flow cytometry. A, Representative flow cytometry analysis of acidic vesicular organelle (AVO) formation in wildtype DLM8 cells. B, Graph representation of CPT-induced AVO formation in wildtype DLM8 and K7M3 cells. *, p < 0.05, compared with same treatment group. C, LC3I/LC3II protein expression. Following 48 h CPT treatment, cells were lysed and cell lysate immunoblotted for LC3I/LC3II protein expression. Increased LC3II expression is indicative of autophagy induction. The expression of treatment group LC3II/actin ratio was determined by densitometry and compared to control group LC3II/actin ratio which was normalized to the arbitrary value of one. Treatment group LC3II expression was normalized to control actin levels as needed. 30ug of protein were loaded for DLM8 LC3I/LC3II determination while only 7.5ug of protein were loaded for K7M3 LC3I/LC3II determination. Actin served as a protein loading control. Data represents the results of three independent experiments, ± SE. p < 0.05 was considered significant. Immunoblot is representative of immunoblots from three independent experiments.