Figure 3:

Ac-α-syn(A53T) seeding of α-syn sensor cell lines is efficient and aggregation is cytotoxic. A) Flow cytometry reports on seeding efficiency of Ac-α-syn(A53T) preformed fibrils for the α-syn(A53T)-CFP/YFP HEK293T biosensor cell line after 24 hours incubation. B) Quantification of cytometry experiments, p = 0.0001, paired t-test of fibril-treated versus lipofectamine treated cells. Error bars are ± S.E.M; n=4. C) Cells harboring aggregates do not propagate. Cultured FRET-negative cells after exposure to buffer (yellow), lipofectamine (orange), or lipofectamine and fibrils (blue) grew to confluency after 4 days while FRET-positive cells exposed to lipofectamine and fibrils (pink) did not. Data are average ± standard deviation; n=3. D) The population of cells in early apoptosis as assessed by Annexin V and PI staining increases with increasing incubation time. Data are average ± standard deviation; n=4. E-H) Colocalization of electroporated Alexa 647–tagged Ac-α-syn(A53T) with Ac-α-syn(A53T)-CFP in biosensor cells after seeding. Exogenously produced Ac-α-syn(A53T) labeled with Alexa fluor 647 (AF⁶⁴⁷) was electroporated into the biosensor cells (pink) and, upon exposure to Ac-α-syn(A53T) fibrils, colocalized with the endogenously expressed Ac-α-syn(A53T)-CFP aggregates (cyan) (Pearson’s coefficient = 0.62).