Figure 3.

Cultures expressing different levels of α-syn respond to nutrient deprivation differently in the expression of markers of ER stress response, autophagy and apoptosis. 3D5 cells differentiated with RA were induced for 0, 3, 6, 9, 12 days to express different levels of α-syn before exposed to media with or without nutrient deprivation for 24 h. (A) Cell lysates were probed with antibodies to α-syn, LC3, GRP78, caspase 3 and β-actin for evaluation of α-syn aggregation, autophagy activation and ER stress response. Molecular weight standards were included as references. Duplicated cultures were included for cell viability assessment. (B–F) Bar graphs summarized the results of quantitative analyses of immunoreactivities of various proteins in cell lysates from three independent experiments with normalization against β-actin immunoreactivities. The average values of Tet (+) group were set as 100%. (G) Calcein assay showing nutrient deprivation caused a significant decrease of cell viability in cells with 9–12 days, but not shorter periods of α-syn induction. Error bars represent standard error of the mean (* p < 0.05, ** p < 0.01, comparing to Tet (+); ^# p < 0.05, ^## p < 0.01, comparing subsets linked by line, n = 3).