Figure 3

HNE-induced cytotoxicity in WT and H50 mutant aSyn overexpressing cells. A) Expression of aSyn in H4 cells transiently transfected with WT aSyn and H50 mutants (H50Q/R) was confirmed by ICC using an antibody against human aSyn (green) and counterstaining by DAPI (blue). B) Transfection efficiency and C) aSyn expression levels in H4 cells transfected with WT aSyn and H50 mutants were assessed via flow cytometry at the time point of HNE treatment (24 h posttransfection). WT and H50Q/R aSyn transfected cells show an equal proportion of aSyn overexpressing cells (B) as well as comparable aSyn expression levels (C). Significance was determined by one way ANOVA Tukey’s Multiple Comparison Tests. n = 3. Error bars = SEM. D) H4 cells transiently transfected with mock, WT, and H50Q/R aSyn were treated with different HNE concentrations (20 – 60 μM) for 12 h and cellular viability was measured by MTS assay. HNE induces a dose-dependent reduction of cell viability. H50Q/R mutant aSyn transfected cells appear to be more resistant to HNE than WT aSyn transfected cells. Significance was determined by two way ANOVA Bonferroni Multiple Comparisons. n = 8. Error bars = SEM.