Figure 7: Progressive ERAD substrate accumulation in the ERQC before appearance of visible Htt aggregates.

(a) The drop in Htt synthesis using TEToff regulation was verified by metabolic labelling for 30 min with [35S]-(Met+Cys) and IP of Htt96Q-Cherry expressed in STHdhQ7/7 cells following incubation with Dox for the indicated periods. (b) Live imaging of a representative STHdhQ7/7 cell expressing Htt96Q-Cherry and H2a-GFP, after shut-off of Htt96Q-Cherry synthesis. Arrow: appearance of a visible large Htt96Q-Cherry aggregate. Arrowhead: H2a-GFP juxtanuclear accumulation. Scale bar, 10 μm. The corresponding movie can be seen in Supplementary Movie 1. (c) H2a-GFP accumulation in the ERQC relative to Htt96Q-Cherry aggregation (from seven independent experiments). Zero: first appearance of a visible Htt96Q-Cherry aggregate. Each line colour represents the same individual cell in both graphs. (d) As in c, but in cells with the presence of visible Htt96Q-Cherry aggregates and H2a-GFP accumulation from the beginning of the observation, (from six independent experiments).