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Fig. 4 Removal of high molecular weight (HMW) ataxin-3 by sodium butyrate is mediated by autophagy. (A) Western blots were performed on protein lysates of SH-SY5Y cells stably expressing ataxin-3-84Q treated with sodium butyrate (SB, 3 mM) and/or 3MA (5 mM) for 72 h and probed for ataxin-3 and LC3B. (B) Quantification of HMW ataxin-3 indicated that SB treatment reduced the amount of HMW ataxin-3, compared to vehicle treatment (p = .0070), and addition of 3MA treatment alone or as a co-treatment prevented the removal of the HMW ataxin-3 (p = .0086 and p = .0079, respectively, n = 6). (C) Quantification of the ataxin-3 monomeric species increased with 3MA treatment compared to the vehicle control (p = .0036). SB+3MA co-treatment further increased ataxin-3 levels compared to vehicle, SB treatment alone, and 3MA treatment alone (p < .0001, p < .0001, and p = .0214, respectively, n = 6). (D) Quantification of the LC3II/LC3I ratio revealed an increased ratio with SB treatment (p = 0002) and the addition of 3MA alone or SB+3MA co-treatment prevented this decrease in LC3II/LC3I (p = .0176 and p = .0098, respectively; n = 6–7). Data represent mean ± SEM. Statistical analysis performed was a two-way ANOVA followed by a Tukey post hoc analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.