Fig. 4

Both UPS and autophagy pathway were involved in mHTT clearance by J3. A–C CFP-103Q-HeLa cells were treated with 20 μM of J3 for 48 h. 10 μg/mL of CHX was added at the last 18 h. The protein expression of mHTT (anti-GFP, sc-9996) was detected by western blot (A), and the quantification of insoluble mHTT (B) and soluble mHTT (C) was analyzed. D–F CFP-103Q-HeLa cells were treated by 20 μM of J3 with or without 10 μM of CQ for 48 h. The protein expression of mHTT (anti-GFP, sc-9996) was detected by western blot (D), and the quantification of insoluble mHTT (E) and soluble mHTT (F) was analyzed. G–I CFP-103Q-HeLa cells were treated with 20 μM of J3 for 48 h. 5 μM of MG132 was added at the last 18 h. The protein expression of mHTT (anti-GFP, sc-9996) was detected by western blot (G), and the quantification of insoluble mHTT (H) and soluble mHTT (I) was analyzed. (J) HeLa cells stably expressing CFP-103Q (Cyan) were treated with J3 for 12 and 24 h, followed by immunostaining of endogenous Ub (Red) and LC3 (Green). Bar = 10 μm. K–L Quantification of aggregates number (K) and aggregates average size (L) was performed. Data are presented as mean ± sem from three individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns means not significant