Extended Data Fig. 9: mHTT–LC3 linker compounds lowered the mutant ATXN3 protein with polyQ expansion in an allele-selective manner.
From: Allele-selective lowering of mutant HTT protein by HTT–LC3 linker compounds
a, Representative western blots and quantifications of ATXN3 levels in a fibroblast line from a patient with SCA3 treated with the indicated compounds. The lowering of mutant (Q74) but not wild-type (Q27) ATXN3 was observed by treatment of linker compounds tested. b, Quantification of the GFP intensity as an indicator of polyQ–sfGFP (25Q–GFP, 38Q–GFP, 46Q–GFP and 72Q–GFP) protein levels in transfected HEK293T cells treated with the indicated compounds using Incucyte. Reduction of 72Q–GFP, 46Q–GFP and 38Q–GFP but not 25Q-GFP was observed. In a and b, the compound concentrations were 100 nM for 10O5 and AN1, and 50 nM for AN2. Bar plots present mean ± s.e.m., and n indicates the number of independently plated wells. c, SDS–PAGE analysis of polyQ–sfGFP proteins (25Q, 38Q, 46Q, 53Q and 72Q) purified from HEK293T cells. The protein purification methods were similar to those for HTT proteins. d, Binding of 10O5, AN1 and AN2 to sfGFP (GFP) or different polyQ–sfGFP (25Q–GFP, 38Q–GFP and 72Q-GFP) proteins in standard treated capillaries measured by MST, performed and analysed similarly as in Extended Data Fig. 2b. All these compounds interact with 38Q–GFP and 72Q–GFP but not with 25Q–GFP or GFP. e, Association–dissociation curves of surface-immobilized compounds 10O5, AN1 and AN2 with polyQ–sfGFP (72Q, 53Q, 46Q, 38Q and 25Q) proteins. For all association–dissociation curves, vertical dashed lines mark the starts of association and dissociation phases of the binding event. The red dashed curves are fits to a Langmuir reaction model with the fitting parameters listed at the bottom of each plot. No binding signals were observed for 25Q–sfGFP (25Q). f–h, Results of mouse behavioural test performed similarly to those in Fig. 5d–f, except that the mice were injected with saline (0.9% NaCl) with DMSO (110 μg kg−1) or without DMSO. The statistical analysis was performed by two-way ANOVA with post hoc Bonferroni’s tests, and F, P values and degrees of freedom are indicated in the table below each plot. In all panels, data are mean ± s.e.m.
