Supplementation with arginine (Arg) reduces filamentous aggregation and autophagosomal accumulation of α-SYN (SYNfil) in tumor necrosis factor-α (TNF-α)-treated SH-SY5Y cells. Representative immunofluorescence images of SH-SY5Y cells, which were grown to confluence in medium containing 100 pg/ml TNF-α (a, C, E) or 100 pg/ml TNF-α and 1 mM Arg (B, D, F). Staining was performed with mAb SYNfil (green), pAb SYN (red), mAb AM (for HS-anMan, green), a pAb against LC3 (autophagosomes marker, red) and DAPI (for nuclei, blue). Exposure time was the same in all cases. Bar, 20 μm. (G) Whisker plot of the number of SYNfil clusters in individual images from repeated experiments with SH-SY5Y cells grown in the presence of TNF-α (n = 16) or TNF-α and Arg (n = 9). (H) Slot blot assay of SYNfil, SYN and HS-anMan (AM) secretion into the media (total volume, 2.8 ml) of cell cultures grown to confluence in the presence of TNF-α or TNF-α and Arg. Sample volume, 30 μL. The intensity of the controls (no primary antibody added) was subtracted. The error bars indicate S.E., n = 5; **, P < 0.01.