Mixed PFFs induce mitochondrial dysfunction. (A) Representative confocal microscopy images show immunostaining of Cox IV (magenta) and α-syn aggregates (green) in α-syn-HEK293 cells treated with different PFFs. Scale bar = 20 μm. Right: Quantitation of Cox IV fluorescence intensity. (B) Representative western blots immunostained for Cox IV, Bcl-2 and Bax of α-syn-HEK293 cell lysates treated with different PFFs. Right: Quantitation of Cox IV, Bcl-2 and Bax. Values are normalized to GAPDH. (C) Complex I enzyme activity of PFFs-treated mitochondria from SH-SY5Y cells. (D) Representative confocal microscopy images showing the morphology of mitochondria in SH-SY5Y cells treated with different PFFs. Right: Quantification of mitochondrial morphologies. The percentage of cells with different mitochondria was quantified. Data are presented as mean ± SEM. P-values were determined by one-way ANOVA followed by Tukey’s multiple comparisons (A–C) and Kruskal–Wallis test followed by Dunn’s multiple comparisons (D). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.