Fig. 6.

XIE62-1004 and XIE2008 accelerate autophagic clearance of mutant huntingtin protein aggregates (mHTT). a Stimulated degradation of GFP-HDQ103 induced by XIE compounds. HeLa cells transiently expressing GFP-HDQ103 were treated with XIE62-1004 (1004), XIE2008 or rapamycin for 18 h and fractionated into soluble and insoluble proteins in 1% Triton X100, followed by immunoblotting analysis. b Inhibition of inclusion body formation by XIE62-1004. HeLa cells expressing GFP-HDQ103 were treated with 10 μM XIE62-1004 for 18 h and analyzed by immunofluorescent analysis of GFP-HDQ103 and immunostaining of p62. c Inhibition of HDQ103 aggregate formation by XIE62-1004. HeLa cells transiently expressing GFP-HDQ25 or GFP-HDQ103 were treated with 10 μM XIE62-1004 or 2 μM rapamycin for 18 h, followed by filter trap analysis. d Facilitated autophagic clearance of HDQ103 aggregates by XIE compounds. Wild-type and ATG5 ^−/− MEFs transiently expressing GFP-HDQ103 were treated with 10 μM XIE62-1004 (1004), 10 μM XIE2008 (2008) or 2 μM rapamycin for 18 h, followed by soluble and insoluble fractionation and immunoblotting analyses. e Inhibition of HDQ74-GFP inclusion body formation by XIE compounds. Inducible PC12 cells stably expressing EGFP-HDQ74 (mutant Htt) were treated with 1 μg/ml doxycycline for 8 h followed by stimulation with XIE62-1004 (1004), XIE2008, or rapamycin for 18 h and subjected to fluorescence analysis of GFP. Average percentage of cells positive for HDQ74-GFP puncta was calculated by counting 100 cells per experimental condition in each experiment. Data represent the mean (±S.D.) of three independent experiments. Statistical significance was calculated using a one-way ANOVA test (****P ≤ 0.0001). f Enhanced autophagic degradation of GFP-HDQ74 in XIE62-1004 stimulated PC12 cells. Inducible PC12 cells stably expressing EGFP-HDQ23 or EGFP-HDQ74 were treated with 10 μM XIE62-1004 or 2 μM rapamycin for 18 h following induction with 1 μg/ml doxycycline for 8 h and fractionated into soluble and insoluble proteins