Fig. 4: Sulfenic acid-modified SOD1 oligomers induce the fibrillization of human SOD1 in SH-SY5Y cells

SOD1 stable cells treated with 5 μM sulfenic acid-modified SOD1 oligomers (+) (a–d), compared with SOD1 stable cells treated without SOD1 oligomers (−) (e–h). SOD1 stable cells (a–h) were incubated with 0 or 5 μM sulfenic acid-modified SOD1 oligomers for 3 days at 37 °C, fixed, ruptured, stained by thioflavin S (green), subsequently immunostained by anti-FLAG antibody and IgG conjugated with Alexa Fluo-546 (red), and observed with confocal microscopy. SOD1 stable cells treated with 1 μM biotinylated sulfenic acid-modified SOD1 oligomers (+) (i–l), compared with SOD1 stable cells when treated without SOD1 oligomers (−) (m–p). SOD1 stable cells (i–p) were incubated with 0 or 1 μM biotinylated sulfenic acid-modified SOD1 oligomers for 3 days at 37 °C, fixed, ruptured and detected by streptavidin DyLight-405 dye (blue), subsequently immunostained by anti-FLAG antibody and IgG conjugated with Alexa Fluo-546 (red); and observed with confocal microscopy. Aggregates extracted from SOD1 stable cells incubated with 5 μM sulfenic acid-modified SOD1 oligomers (q) for 3 days at 37 °C were labeled with gold particles. Red arrowheads were used to highlight amyloid fibrils. SOD1 fibrils were clearly observed by immunogold electron microscopy in such a case. Sulfenic acid-modified SOD1 oligomers (a–d) were formed when apo wild-type SOD1 (30 μM) was incubated with 100 μM hydrogen peroxide in 20 mM Tris-HCl buffer (pH 7.4) and at 37 °C for 6 h, and then labeled with biotin (i–l). The scale bars represent 10 μm (a–p) and 20 nm (q), respectively