Fig. 4. Fibril seeds from H46R and G85R are more cytotoxic to cultured cells than wild-type SOD1 fibril seeds generated under the same conditions.

(A to F) Cytotoxicity of fibril seeds from H46R and G85R to SH-SY5Y neuroblastoma cells (A and B), HEK-293T cells (C and D), or HEK-293T cells stably expressing FLAG-tagged wild-type SOD1 (E and F) assessed by the CCK8 assay (A, C, and E) and the MTT assay (B, D, and F), compared with that of fibril seeds from wild-type SOD1. Cells were cultured for 1 day and then incubated with 0 μM SOD1 fibril seeds, 10 μM wild-type SOD1 fibril seeds, 10 μM H46R fibril seeds, and 10 μM G85R fibril seeds, respectively, for 1.5 days. The cell viability (%) (open red circles shown in scatter plots) was expressed as the means ± SD (with error bars) of values obtained in n = 5 (A, E, and F) or 6 (B to D) biologically independent experiments. SOD1 fibrils, P = 0.000030, 0.000011, 0.000041, 0.00051, 0.0227, and 0.000014 (A to F); H46R fibrils, P = 0.00000089, 0.0000011, 0.0000045, 0.0000015, 0.000015, and 0.00000023 and 0.012, 0.0033, 0.000063, 0.00022, 0.0074, and 0.00996 (A to F); and G85R fibrils, P = 0.0000010, 0.000011, 0.0000084, 0.000037, 0.0000011, and 0.000000084 and 0.024, 0.045, 0.00026, 0.0048, 0.0046, and 0.026 (A to F). Statistical analyses were performed using two-sided Student’s t test. Values of P < 0.05 indicate statistically significant differences. The following notation is used throughout: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 relative to control. Cells treated with 20 mM tris-HCl buffer (pH 7.4) containing 5 mM TCEP for 1.5 days were used as a control.