Fig. 2: αS fibrils are largely localized at the surface of the cellular membrane but ROS generation correlates with the intracellular αS pool.

a Representative confocal scanning microscope images showing the basal, median, and apical sections of SH-SY5Y cells treated for 1 h with the indicated αS species at 0.3 μM and the median sections of untreated cells. Red and green fluorescence indicates the cell membranes and the αS species revealed with wheat germ agglutinin (WGA) and polyclonal anti-αS antibodies (Ab52168, Abcam), respectively. The histogram on the right reports a semi-quantitative analysis of the intracellular and extracellular αS-derived fluorescence data expressed as the percentage of endogenous αS fluorescence. b Dependence of ROS production on the intracellular αS-derived fluorescences in SH-SY5Y cells treated with αS species. ROS values reported in Fig. S5 were plotted against the αS-derived fluorescence values reported in Fig. S4d of cells treated with OB* and SF at the corresponding concentrations. c Representative confocal scanning microscope images showing mitochondrial superoxide production detected with MitoSOX probe in living SH-SY5Y cells. Red and green fluorescence indicates MitoSOX staining and αS labeled with AF488 dye, respectively (six independent experiments with one internal replicate). d Dependence of mitochondrial superoxide production on the intracellular AF488-derived fluorescence signal in SH-SY5Y cells treated with αS species. Experimental errors are S.E.M. (n = 4 with three internal replicates in panels (a), (b); n = 6 in panel (d) with one internal replicate). Samples were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test relative to untreated cells (in panel a, *P < 0.05, **P < 0.01, ***P < 0.001; in panel b, P < 0.001; in panel d, P < 0.001). A total of 200–250 cells were analyzed per condition.