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.