Figure 7
Increased protein amount and aggregation in αSyn-overexpressing H4_GE cells upon incubation with DMSO and FeCl3. H4_GE-S, H4_GE-V1S + SV2 and H4_GE-V cells were incubated with 0.75% DMSO and 100 µM FeCl3 ( + FeCl3) or 0.1% DMSO (control) for 3 days. (A) Protein amounts increased in cell lines overexpressing variants of αSyn but not in cells overexpressing Venus upon incubation with 0.75% DMSO and 100 µM FeCl3. For quantification signals were normalized to housekeeper. Signals from samples treated with 0.1% DMSO were set to 100%. Bars show mean of 3 to 4 independent experiments and error bars show SEM. Full-length Western blots and additional exposures are shown in Supplementary Figs 9A, 10A, respectively. (B) Representative Western blots from cell lysates after sucrose gradient centrifugation of H4_GE-S (left), H4_GE-V1S + SV2 (middle) and H4_GE-V cells (right) incubated with 0.1% DMSO and 0 µM FeCl3 (top) or 0.75% DMSO and 100 µM FeCl3 (bottom) demonstrating an increase in high molecular weight species in cell lines overexpressing variants of αSyn but not in cells overexpressing Venus following treatment with 0.75% DMSO and 100 µM FeCl3 (n = 3). Full-length Western blots and additional exposures are shown in Supplementary Figs 9B, 10B, respectively. (C) The fraction of condensed nuclei (red arrowheads) was considerably increased in αSyn-overexpressing cells, but only slightly in Venus-overexpressing cells following incubation with 0.75% DMSO and 100 µM FeCl3 suggesting increased cytotoxicity. Scale bar is 50 µm and valid for all panels. Bars show mean of 4 independent experiments, error bars show SEM.
