Fig. 2: Heme minimizes the in-cell seeding, synthetic SUV permeabilization and cytotoxicity of various aggregated species of α-Syn.

a–c α-Syn-EGFP-transfected SH-SY5Y cells were transduced and incubated for 24 h with 1 μM fibril seeds prepared by sonicating preformed α-Syn fibrils that had been incubated under aggregation-inducing conditions a without or b with heme for 96 h (α-Syn/heme = 25:1). Punctate green structures (white arrows) within the cytoplasm in (a, bottom inset) denote seeded α-Syn aggregates. c Cells transfected with α-Syn-EGFP but not transduced with seeds (vehicle) do not convert into an inclusion-positive state. The scale bars denote 10 μm. d–f The effect of α-Syn structural conformers formed after various periods of aggregation d in the absence of heme, or e in presence of heme: where heme was added from the beginning of aggregation period (preincubation), or f when heme was added after various periods of aggregation had occurred (postincubation), on calcein-loaded liposomal SUV permeation (black bars), early apoptosis (red bars) and late apoptosis/ necrosis (blue bars) as observed in SH-SY5Y neuroblastoma cells. For the FACS and calcein release experiments, error bars represent standard error of the mean and have been calculated from six independent experiments, with each condition replicated thrice.