Extended Data Figure 10. Effect of different disease proteins with polyQ expansion on beclin 1 ubiquitination, beclin 1 levels and starvation-induced autophagy.

a, HeLa cells were transfected with empty vector, GFP atrophin-1 (ATN-1) Q19, GFP ATN-1 Q71 and HA Ub for 24 hr, treated in the last 6 hr with proteasome inhibitor (MG132 10 µM). Endogenous beclin 1 was immunoprecipitated from the lysates for ubiquitination analysis and for detection of bound ATN-1 using anti-ATN-1 antibody. b, HeLa cells were transfected with empty vector, HA androgen receptor (AR) Q25, HA AR Q120 and HA Ub for 24 hr, treated in the last 6 hr with proteasome inhibitor (MG132 10 µM). Endogenous beclin 1 was immunoprecipitated from the lysates for ubiquitination analysis and for detection of bound AR using anti-AR antibody. c, Primary fibroblasts derived from healthy controls (n=5) and from HD patients (n=7) were starved with HBSS together with bafA1 (400 nM) for 4 hr and analysed for LC3-II levels. Results are mean ± s.e.m. * P<0.05 one-tailed Mann Whitney test. d-f, Primary fibroblasts derived from healthy controls and from patients with different polyQ diseases were kept in full media or starved with HBSS for 4 hr and analysed for LC3-II levels (LC3-II/actin ratio is presented). d, Spinocerebellar ataxia type 3 (SCA3) samples. e, Dentatorubral-pallidoluysian atrophy (DRPLA) samples. f, HD samples. The BafA1 experiments for these sets of patients are presented in Fig. 4. g, Beclin 1 levels (beclin 1/actin ratio is presented) in the starved control cells vs. SCA3, SCA7, DRPLA and HD patient cells. We only had one SCA7 patient sample and thus we have not analysed it further.