Transfection and Differentiation of Rat and Human Neuronal Cell Lines

Undifferentiated ST14A cells, a rat striatal progenitor cell line with high transfectability and many neuronal characteristics (gift from Dr. Elena Cattaneo, Milan IT), were cultured according to standard protocols [22 (link)]. 2 μg of total DNA was transfected per well in 6 well plate. JetPEI DNA transfection reagent (Polyplus Transfection Inc.) was used to transiently transfected cells as previously described [23 (link)]. For all transfections unless otherwise noted, cells were imaged 48 hours’ post-transfection, and then harvested for downstream applications. For endogenous α-Syn assay, normal SH-SY5Y cells (ATCC) were transiently transfected using Jet PRIME DNA transfection reagents (Polyplus Transfection Inc.), with control (CON; empty vector) or VH14, VH14PEST, or VH14SCRPEST (scrambled inactive PEST) constructs. They were then differentiated into the neuronal pathways 48 hours post- transfection for 3 days with 10 μM retinoic acid (RA) prior to harvesting [24 (link)]. To verify that targeted degradation is occurring through the proteasome, specific proteasome inhibitor epoxomicin (10 μM per well) or the vehicle DMSO was applied to the cells 12 h prior to the harvest in a subset of experiments.