Figure 2.

Cells with 10 days of α-syn induction differ from those without induction in response to nutrient deprivation. RA-elicited differentiated 3D5 cells with or without TetOff induction for 10 days, regarded as Tet (−) and Tet (+), were respectively subjected to nutrient deprivation for 24 h. Sibling cells without nutrient deprivation were used as Controls (Con). (A) Cell lysates were probed with antibodies to α-syn, LC3, p62, Beclin-1, p-mTOR, T-mTOR, Caspase 12, Lamp2, GRP78, p-EIF2α, T-EIF2α, CHOP, cleaved Caspase 3, SREBP1, SREBP2 and β-actin by western blotting. Molecular weight standards were included as references. Duplicate cultures were included for cell viability assessment. (B–O) Bar graphs summarized quantitative analysis of the immunoreactivities of interesting proteins from three independent experiments and normalized with β-actin or T-EIF2α immunoreactivities. The average values of Tet+/Con group were set as 100%. (P) Calcein assay showed that nutrient deprivation caused a significant lose of viability in cells overexpressing α-syn, but not in those expressing endogenous α-syn. (Q,R) Quantitative real-time polymerase chain reaction showed that nutrient deprivation did not change the transcripts level of α-syn and LC3 in 3D5 cells with and without α-syn overexpression (p > 0.05; n = 3). (S) The assay showed that nutrient deprivation did not change the activity of Cathepsin D in 3D5 cells with and without α-syn overexpression (p > 0.05; n = 3). Bar graph summarized the data from three independent experiments. Error bars represent standard error of the mean (* p < 0.05, ** p < 0.01, comparing to Tet+/Con; ^# p < 0.05, ^## p < 0.01, comparing subsets linked by line; N.S., no significant difference comparing subsets linked by line).