Figure 4

Autophagy inhibition has an opposing impact on CPT-induced cell death. A, ATG5 protein levels in DLM8 and K7M3 cells following shRNA-mediated knockdown of ATG5. Cells were infected with lentivirus containing empty shRNA vector or lentiviral shRNA targeted against ATG5 mRNA. Following infection, cells were lysed and total protein collected. To confirm ATG5 protein knockdown and autophagy inhibition, total protein was immunoblotted for ATG5 and LC3I/C3II protein levels, respectively. Actin served as a protein loading control. B, Acidic vesicular organelle (AVO) formation. Autophagy-competent and autophagy-inhibited DLM8 cells were treated with CPT for 24 h followed by assessment of AVO formation. Impact of autophagy inhibition on cell death in C, DLM8 and D, K7M3 OS cells. Autophagy-competent and autophagy-inhibited DLM8 and K7M3 cells were treated with CPT as indicated for 48 h. Following drug treatment, cell viability was assessed by trypan blue exclusion. *, p < 0.05, compared with same treatment group. E, Basal levels of autophagy in MC3T3, DLM8 and K7M3 cells. Cells were untreated and allowed to grow to approximately 70% confluency. Cells were then collected, lysed and total protein immunoblotted for LC3I/LC3II. 30ug of protein were loaded for each cell line. Actin served as a protein loading control. F, Phosphorylation of p53 in DLM8 cells. Cells were treated with CPT as indicated for 24 h. Following CPT treatment, cells were lysed and cell lysate probed for phospho p53 and total p53 protein expression. Data represents the results of at least three independent experiments, ± SE. p < 0.05 was considered significant. Immunoblots are representative of immunoblots from two independent experiments.