Figure 3

Ebselen and its derivatives reduce aggregating species in human cell model. (A) Effects of drug (MR6-26-2) doses 25, 50 and 100 µM on SOD1 dimerization following transfection and drug treatment of H4 cells with A4V (V_N + V_C), scale bar: 25 µm. Left to right panel for each dose shows transmitted light (TL), DRAQ5 stained nuclei, SOD1 VFP (after complementation of SOD1 V_C or SOD1 V_N) and merged images respectively. (B) Quantification of the number of inclusions per cell. 50–200 cells were counted per condition and per experiment and classified into three groups: blue, orange, and grey bars represent the percentage of cells without inclusions, with 5 or less inclusions and cells with more than 5 inclusions respectively. Data are expressed as mean ± SD of at least three independent experiments. One-way ANOVA, with Dunnet’s multiple comparison, was used for statistical analysis with significance level of * p < 0.05 ***p < 0.001 or ****p < 0.0001, which represents statistically different results between mutant SOD1 with and without compound treatment. (C) Representative images showing reduction in the aggregation propensity of H4 cells expressingA4V, G93A, H46R and D90A following treatment with most effective MR6-26-2 compound versus non-treated cells (scale bar: 50 µm). White arrows indicate the mutant SOD1 aggregation. The scaling of the images has been kept different to make the aggregates/inclusion bodies clearly visible in non-treated conditions, and to show the maximum possible number of cells within a single field as proof of drug efficacy in stable SOD1 dimerization and uniform fluorescence following treatment with MR6-26-2.