Figure 5

MPP+-induced cell death is inhibited by ferroptosis inhibitors, but not by CsA. (a) Neuronal SH-SY5Y cells were treated for 48 h with MPP+ (5 mM) in the presence of a CypD-dependent cell death inhibitor (CsA, upper left), ferroptosis inhibitors (Fer-1, upper right; DFO, lower left; Trolox, lower right) at the indicated concentrations. Cell death was calculated from lactate dehydrogenase (LDH) leakage. (b) Representative C11-BODIPY staining of lipids in the membrane of WT neuronal SH-SY5Y cells. Red signals indicate non-oxidized lipids and green signals indicate oxidized lipids. Cells were treated with MPP+ (5 mM) in the presence of Nec-1 (20 μM), DIM (20 μM), Fer-1 (2 μM), DFO (100 μM), or dimethyl sulfoxide (DMSO) (control) for 48 h, and subjected to fluorescence microscopy. Scale bar=50 μm. (c) Red and green signals in (b) were quantified by using the Image J software. The lipid peroxidation ratio was calculated as follows: mean value for green signals/(mean value for red signals+mean value for green signals). Each cell field was selected by the minimum error algorithm. (d) WT and p53-deficient neuronal SH-SY5Y cells were treated with MPP+ in the presence or absence of Fer-1 (2 μM) for 48 h. Cell death was calculated from LDH leakage. (e) Representative C11-BODIPY staining of lipids in membrane of p53-deficient neuronal SH-SY5Y cells. Red signals and green signals indicate non-oxidized and oxidized lipids, respectively. Cells were treated with MPP+ (5 mM) in the presence of Nec-1 (20 μM), DIM (20 μM), Fer-1 (2 μM), DFO (100 μM), or DMSO (control) for 48 h, and subjected to fluorescence microscopy. Scale bar=50 μm. (f) Red and green signals in (e) were quantified by using the Image J software. The lipid peroxidation ratio was calculated as follows: mean value for green signals/(mean value for red signals+mean value for green signals). Each cell field was selected by the minimum error algorithm. Data are shown as the mean±S.D. of three independent experiments. **P<0.01; ***P<0.001.