Fig. 6.

Role of p97 and UBXN1 in Huntingtin polyQ aggregation. (A) U2OS cells were treated with doxycycline to induce the expression of HTT Q91–mCherry. Cells were fixed and stained for ubiquitin and UBXN1 to demonstrate colocalization with HTT Q91–mCherry. (B) UBXN1 was transiently depleted with siRNAs in U2OS HTT Q91-mCherry cells and imaged for inclusion body formation. The percentage of cells with HTT Q91–mCherry inclusion bodies was quantified. (C) PuLSA analysis of HTT Q91-mCherry aggregates in UBXN1- and p97-depleted cells. (D,E) Representative fluorescent images of L4 larvae stage wild-type, cdc-48.1(tm544) (D), ubxn-1(tm2759) and ubxn-4(ok3343) worms. (E) Loss-of-function mutants expressing polyQ40::YFP in body wall muscle. Images were taken of worms precisely age-matched at the L4.4 vulva developmental stage. Bottom panels in each show quantification of visible fluorescent aggregates in L4 larvae animals expressing polyQ40::YFP in wild-type, cdc-48.1, ubxn-1 and ubxn-4 mutant animals. Quantification was only performed on worms at the L4.4 vulva development stage based on vulva morphology. The black dot represents the mean from each biological replicate. The indicated number of cells (n) analyzed from all three independent biological replicates is shown for each condition. Graphs show mean±s.e.m. *P≤0.05, **P≤0.01, ****P≤0.0001 as determined by one-way ANOVA with Tukey post-hoc testing (B) or Dunnett's test (E), and unpaired Student's t-test (D). Scale bar: 10 µm. Scale bars: 10 μm (B); 100 μm (D).