Fig. 1

Autophagy activation by trehalose in neuro-2A cells expressing mutant ataxin-3. Neuro-2A cells expressing mutant ataxin 3 (MutAtx-3) were treated with 10 mM trehalose or vehicle (control) from 0.5 to 72 h (h). Protein levels of LC3BI, LC3BII, endogenous ataxin-3 (EndAtx-3) and mutant ataxin-3 (MutAtx-3) were quantified by western blot. a Representative picture of Western blot. b Quantitative densitometric analysis of western blot bands. Values are presented as mean ± SEM (n = 4–5). Results are expressed as % of control. *^{,#, $}p < 0.05; **^{,##, $$}p < 0.01, compared to control. One-way analysis of variance (ANOVA), followed by a Dunnett’s post hoc analysis for multiple comparisons; outliers were excluded using the Grubb’s test. Cells were then incubated for 72 h with trehalose 10 mM, in the presence and absence of the lysosomal degradation inhibitor, chloroquine, for 6 h before protein collection. Protein levels of LC3BII were quantified by western blot. c Representative picture of Western blot. d Quantitative densitometric analysis of western blot bands. Values are presented as mean ± SEM (n = 6). Results are expressed as % of control. ^##p < 0.01, compared to control; Student’s t test. ****p < 0.0001, compared to control; One-way analysis of variance (ANOVA), followed by a Dunnett’s post hoc analysis for multiple comparisons. e Autophagic flux quantification by the LC3BIIturnover assay. Difference between densitometric intensity of LC3II bands between control and trehalose conditions in the presence and absence of lysosomal inhibitors. Values are presented as mean ± SEM (n = 6). ⁺p<0.05, compared to control plus chloroquine; Student’s t test