Extended Data Fig. 7: mHTT–LC3 linker compounds did not influence autophagy.

a, HeLa cells stably expressing GFP–LC3B were treated with 2 μl vehicle (0.1% DMSO), 10O5 or AN2 for the indicated concentration for 24 h; chloroquine (CQ, 20 μM) treatment was used as a control. After 24 h, cells were fixed and images were acquired by confocal microscopy. The number and size of GFP vesicles per cell was determined using ImageJ software (n indicated on top of each plot). For each treatment, more than 20,000 puncta were quantified (~100 puncta per cell from 226 cells). Scale bar, 10 μm. b, Representative images and quantifications of the numbers of autophagosomes (GFP⁺ puncta) and autolysosomes (RFP⁺GFP⁻ puncta) in HeLa cells stably expressing mRFP–GFP–LC3B. Scale bar, 10 μm. Autophagosome numbers or sizes were not influenced by 10O5 and AN2 at the indicated concentrations after 24 h treatment (or 4 h treatment, not shown). The autophagsome fusion was also unaffected as indicated by the autolysosome number. Note that the autophagosome and autolysosome numbers and sizes were based on image analysis of the puncta, some of which may represent multiple vesicles. Green vesicles are considered to be autophagosomes (GFP⁺ puncta) and red vesicles are considered to be both autophagosomes and autolysosomes. The number of autolysosomes (RFP⁺GFP⁻ puncta) was calculated by subtracting the number of green vesicles from that of the red vesicles. More than 10,000 puncta from 194 cells were analysed. c, Representative western blots and quantifications of HeLa cells stably expressing GFP–LC3B. The ‘free GFP’ was generated by lysosomal cleavage, and thus the free GFP/GFP–LC3B ratio was used as an index for autophagy flux, which was unaffected by 10O5 or AN2, but decreased by the autophagy flux inhibitor chloroquine. d, Representative western blots and quantifications of the chase signal of long-lived proteins in HeLa cells as an indicator of autophagy flux (see Methods). Consistent with previous reports³³, starvation reduced the long-lived protein chase signal, whereas rapamycin treatment had a milder effect. The mHTT–LC3 linker compounds 10O5 and AN2 had no influence in this assay. e, Representative western blots and quantifications of LC3 in cultured cortical neurons treated with the indicated compounds. Normalized LC3-II/LC3-I was used as the indicator of autophagy. Right blot: 10O5, 100 nM; AN2, 50 nM. f, SQSTM1 (p62) levels were determined by western blot for the cortical tissues from mice injected with the indicated compounds or DMSO control. Bars indicate mean and s.e.m.; n indicated in each bar shows the number of cells (a, b), the number of independently plated wells (c–e) or the number of mice (f). Data are mean ± s.e.m. The statistical analysis was performed by one-way ANOVA with post hoc Dunnett’s tests (a–e) or two-tailed unpaired t-tests (f). Note that the post hoc tests were not performed if the ANOVA tests failed to show significance. ****P < 0.0001 (post hoc test).

Source data