a, Representative western blots (HTT detected by the 2166 antibody) and quantifications of compound-treated cultured cortical neurons from HdhQ7/Q140 HD-knock-in mice. The neurons were treated with the indicated compounds (100 nM for 10O5, 8F20 and AN1; 50 nM for AN2) with or without the autophagy inhibitor NH 4 Cl (top) or chloroquine (bottom left), or the autophagy activator rapamycin (bottom right). The same amount of culture medium was added in the controls (top). The statistical analysis was performed by one-way ANOVA with post hoc Dunnett’s tests, and the F, degree of freedom and post hoc P values are indicated in each bar plot. b, Western blots using indicated HTT or polyQ antibodies for samples from cultured cortical neurons treated with the indicated compounds: 10O5 (100 nM), 8F20 (100 nM) or AN2 (50 nM). The HTT gel blots presented in Fig. 2d (right) were cropped from first four blots. The low molecular weight bands were run out in these blots so that the wtHTT and mHTT could be better separated. Note that the weak bands just above 250 kDa in the first two blots were leftover signals from the spectrin blotting. The spectrin signals were too strong to be stripped completely. c, Western blots using the antibody MW1 or 3B5H10, which detects mHTT specifically. We ensured that the relatively low-molecular-weight proteins did not run out of the gels. No increase of potential polyQ-containing mHTT N-terminal fragments was observed. d, iPS-cell-derived striatal neurons from a patient with HD (Q47) were treated with the indicated compounds (100 nM, with 0.1% DMSO) in presence of an additional 0.1% DMSO or 10 mM NH 4 Cl, and the mHTT levels were measured by HTRF using the 2B7/MW1 antibody pair. All signals were normalized to the averaged signals from the DMSO control group. The statistical analysis was performed by one-way ANOVA with post hoc Dunnett’s tests, and F, degree of freedom and post hoc P values are indicated in each bar plot. ****P < 0.0001. The post hoc analysis was not performed if the ANOVA tests did not show significance (P > 0.05). e, Immortalized fibroblasts from a patient with HD (Q47) were transfected with the non-targeting control siRNA (Neg siRNA) or the ATG5 siRNA (target sequence, GCCUGUAUGUACUGCUUUA; ATG5 mRNA was knocked down to 17.7 ± 3.0%, n = 3, as tested by reverse transcription with quantitative PCR), and then treated after 24 h with the indicated compounds (100 nM) for a further 48 h. mHTT levels were then measured by HTRF using the 2B7/MW1 antibody pair. All signals were normalized to the averaged signals from the DMSO control group. The statistical analysis was performed by one-way ANOVA with post hoc Dunnett’s tests, and F, degree of freedom and post hoc P values are indicated in each bar plot. ****P < 0.0001. The post hoc analysis was not performed if the ANOVA tests did not show significance (P > 0.05). The western blot of LC3 confirmed the partial inhibition of autophagy in the ATG5-knockdown cells. f, Similar to e, but in wild-type (WT) or Atg5-knockout (Atg5 KO) mouse embryonic fibroblast lines (MEF) transfected with full-length mHTT (flHTT-Q73). The western blot of LC3 confirmed the inhibition of autophagy in the Atg5-KO cells. For all panels, n indicates the number of independently plated wells, and bars represent mean and s.e.m. Full-blots of cropped gels are shown in Supplementary Fig. 1. Source data