FIG. 4.

The Vps34 inhibitor 3-methyl adenine prevents H₂O₂ toxicity in SH-SY5Y cells. (A) SH-SY5Y cells plated on coverslips were exposed to 200μM H₂O₂ (perox) for 60 or 120 min and then fixed and processed for nuclei staining with propidium iodide (PI) and immunofluorescence detection of LC3-positive autophagosomes; 35 ± 5% of LC3-positive cells showed chromatin alteration typical of apoptotic cell death. (B) SH-SY5Y cells adherent on coverslips were preincubated or not with 3MA and exposed or not to 200μM H₂O₂ (perox) for 2 h. At the end, the cells were processed for nuclei staining with 4′,6-diamidino-2-phenylindole (DAPI) and immunofluorescence detection of LC3-positive autophagosomes. The arrow points to chromatin condensation and fragmentation in peroxide-treated cells. Bar = 10 μm. (C) Adherent cells preincubated or not with 3MA and exposed or not to 200μM H₂O₂ (perox) for 2 h. At the end, the cells were processed for cytofluorometry analysis of annexin V-FITC–labeled cells. Data (means ± SD) of four independent experiments. (D) Clonogenic assay to demonstrate the long-term protection by 3MA against peroxide stress. Two sets of cultures pretreated or not with 3MA were incubated for 4 h with or without H₂O₂ (perox), as indicated. At the end, the adherent viable cells of one set of cultures were counted. The cells of the other set of cultures were rinsed and cultivated for further 40 h in fresh medium and at the end the viable cells were counted. Cell density data (±SD) of two experiments in triple. (E) Cytofluorometric analysis of annexin V-FITC–positive cells of the cultures treated as above.