Fig. 4. Overexpressed SPY1 resists ferroptosis in the cell model of ALS.
A The effect of SPY1-Flag or SiSPY1 transfection in hSOD1G93A or NSC34 cells was evaluated by Western blotting. B Microscopic images were used to show response to RSL3 (2 μM for 2 h) of NSC34 cells with SPY1-Flag and Flag. Scale bar, 100 μM. C The effect of overexpressed SPY1 on ferroptosis induction was assessed by LDH assay in NSC34 cells with the treatment of diverse dosages of RSL3 for 2 h (n = 6). D, E Images and quantification of C11-BODIPY fluorescence exhibited the level of lipid oxidation with the increase of transfected SPY1-Flag in hSOD1G93A cells (n = 3). Scale bar, 100 μM. F, G The effect of increased SPY1 on relative LIP and GSH was shown (n = 6). H The lipid peroxidation of inner mitochondria membrane was measured by MitoPeDPP immunofluorescence staining followed by flow cytometry (channel 488, n = 3). I The changes in mitochondrial membrane potential caused by mutant SOD1 and overexpressed SPY1 were detected by JC-1 fluorescence staining followed by flow cytometry. J–L The effect of overexpressed SPY1 on RSL3-induced ferroptosis in SHSY5Y, Hela, and HEK293T cells was measured by LDH assay (n = 6). Values represent mean ± SD. Statistical analysis by one-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.