Figure 5.

ULK1 exerts an essential role in autophagy-mediated mutATXN3 clearance in vitro

(A) Schematic representation of the experimental conditions. Human ULK1 KO and control HAP1 cell lines were infected with lentivirus encoding for mutATXN3 and ULK1. (B) The stable expression of the transgenes was confirmed by western blot analysis. (C) HAP1 cells were analyzed in the presence or absence of chloroquine (100 μM). Western blot analysis of LC3B shows a significant decrease of LC3BII net flux in the ULK1 KO cell line that trended toward recovery upon ULK1 overexpression. (D) Western blot analysis of ATXN3 shows a significant decrease of mutATXN3 levels in HAP1 KO expressing ULK1. Densitometric quantification of LC3BII levels and mutATNX3 levels relative to β-actin and β-tubulin, respectively. Values are expressed as mean ± SEM of n = 4. A one-way ANOVA with Bonferroni’s post hoc test was used to evaluate statistical significance (∗p < 0.05, ∗∗p < 0.01). (E) Schematic representation of Neuro2a transfection with plasmids encoding for the mutATXN3 with 69Q and GFP, ULK1, or ULK1 + ULK2, using PEI reagent. (F–H) Western blot analysis shows a significant reduction of high-MW species (G) and soluble mutATXN3 levels (H). Densitometric quantification of mutATXN3 levels relative to β-actin. Values are expressed as mean ± SEM of n = 4 and 5. A one-way ANOVA with Dunnett’s post hoc test was used to compare Neuro2a cells expressing ULK constructions to GFP (∗p < 0.05, ∗∗p < 0.01). CTRL NI, non-infected control; CTRL, control; KO, knockout; chQ, chloroquine; PEI, polyethyleneimine; MW, molecular weight