Fig. 4
PACER overexpression in NSC34 cells increases SOD1 aggregation and impairs autophagy. A–C NSC34 cells were transiently co-transfected with expression vectors for human wild-type SOD1 (SOD1WT) or mutant SOD1G85R or SOD1G93A all fused to EGFP together with a vector for mouse or human V5-tagged Pacer or PACER, respectively, or a mock control. After 48 h, SOD1 levels under reduced (+DTT) and non-reduced (−DTT) conditions were monitored in cell extracts prepared in 1% Triton X-100 buffer by western blot. Representative blots of 3 experiments are shown. B Filter trap assay (n = 3), and C densitometric quantification of the filter trap assay of SOD1 mutants and wild-type (n = 3). Mean and SEM are shown. D and E Autophagy flux was performed in NSC34 cells transiently transfected with a plasmid for human V5-tagged PACER or plasmid vector alone (Mock) and treated with EBSS and/or lysosome inhibitors (Lys. Inh.: Bafilomycin A1, E64D, and leupeptin) at different time points (0.5 h, 2 h, 4 h). D Cell extracts were subjected to western blot. LC3B-II, p62, Beclin1 and V5 expression were verified. β-Actin levels were used as a loading control (n = 3). E Autophagy flux was quantified by densitometric analysis of LC3-II (n = 3) normalized to β-Actin. Mean and SEM are shown. In C and E statistical analysis was performed using One-way ANOVA. p values: n.s., non-significant, *, p ≤ 0.05; **, p ≤ 0.01;***, p ≤ 0.001