Fig. 4
From: Network approach identifies Pacer as an autophagy protein involved in ALS pathogenesis
Depletion of Pacer impairs autophagosome formation and promotes SOD1 aggregation. a Autophagy flux under Pacer knockdown. Cells were treated with EBSS medium or/and lysosome inhibitors (Lys. Inh.) for 0.5, 2 and 4 h. Cell extracts were subjected to Western blot. As a mock control, a scrambled shRNA (shCtrl) construct was used. Pacer, Beclin1, p62 and LC3-II formation levels were determined. β-Actin serves as a loading control. b Densitometric quantifications of LC3-II flux (n = 3). One-way ANOVA and Bonferroni’s post hoc tests were performed.Mean and SEM with only statistically significant p-values are shown: *, p ≤ 0.05. c-f, NSC34 cells depleted of Pacer were transiently co-transfected with expression vectors for human wild-type or mutant SOD1G93A fused to EGFP. When indicated, human Pacer (hPacer-V5) was co-expressed. c and d, after 48 h, SOD1 aggregation was assessed under non-reducing (−DTT) conditions. Cell extracts were prepared in 1% Triton X-100 buffer or 1% SDS buffer for Western blot and filter trap assays, respectively. In c HSP90 serves as a loading control. e SOD1 inclusions in NSC34 cells were assessed by confocal microscopy. Percentages of cells with SOD1WT-EGFP or SOD1G93A-EGFP inclusions are shown. f Percentage of cell death was quantified at 72 h (SytoxBlue positive, SB+) in NSC34 stable lines expressing shPacer or shCtrl transiently transfected with EGFP, SOD1WTor SOD1G93A, and hPacer-V5. In e and f statistical analyses were performed using one-way ANOVA and Bonferroni’s post-hoc tests. Mean and SEM with only statistically significant p-values are shown: *, p ≤ 0.05; **, p ≤ 0.01; and ***, p ≤ 0.001