Figure 5

Autophagy inhibition decreases CPT-induced oxidative stress and buthionine sulfoximine (BSO)-induced cell death. Cells were treated with CPT for 24 h followed by incubation with HE or DCFH-DA. A, Camptothecin-induced ^.O₂ ^-. *, p < 0.05. B, Camptothecin-induced H₂O₂. *, p < 0.05. C, Autophagy-competent cells are more sensitive to BSO-induced cell death. Cells were pretreated with 1mM BSO for 2 h followed by 48 h CPT treatment. Cells received a second 1mM BSO treatment 12 h into the CPT treatment. Following CPT treatment, cell viability was determined by trypan blue exclusion. *, p < 0.05, compared with control group. D The antioxidant NAC reverses BSO-induced cell death. Cells were pretreated with NAC for 2 h prior to BSO treatment. Cells received a second 1mM BSO treatment 12 h into the BSO treatment. Following 48 h BSO treatment, cell viability was determined by trypan blue exclusion. *, p < 0.05, compared with control group. E, BSO treatment increases LC3II levels in autophagy-competent DLM8 cells. Cells were treated with 1mM BSO for times indicated in figure. F, NAC pretreatment inhibits CPT-induced autophagy induction in autophagy-competent DLM8 cells. Cells received no drug, NAC, CPT or combination as indicated in figure for 48 h. For combination groups, cells were pretreated with NAC for 2 h. G, NAC pretreatment inhibits BSO-induced autophagy induction in autophagy-competent DLM8 cells. Cells received no drug, NAC, 1mM BSO or combination for 6 h. For combination group, cells were pretreated with NAC for 2 h. Following drug treatment, cells were lysed and total protein immunoblotted for LC3I/LC3II protein expression. Actin served as a protein loading control. Data represents the results of three independent experiments, ± SE. p < 0.05 was considered significant. Immunoblots are representative of immunoblots from at least two independent experiments.