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Fig. 1. Rab5 modulates the aggregation and toxicity of mutant huntingtin. (a) Quantification of GFP-expressing COS-7cells showing signs of cell death that had been transiently transfected with dominant-negative (DN), constitutive active (CA) or wild-type (WT) Rab5, or empty vector control, and the EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats (Q74) (at a 3:1 ratio) for 48 hours. ^***P<0.0001, ^**P<0.001, ^*P<0.05. (b) Quantification of GFP-expressing COS-7 cells containing aggregates that were quantified for toxicity shown above. ^***P<0.0001. (c) Quantification of GFP-expressing HeLa cells containing aggregates transiently transfected with siRNA targeting Rab5a, Rab5b, Rab5c or all three siRNA simultaneously (Rab5abc) for 72 hours, and also with EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats for the last 24 hours of the 72-hour siRNA transfection period. ^***P<0.0001, ^*P<0.05. (d) Rab5 overexpression increases the numbers of rhabdomeres in ommatidia of mutant huntingtin-expressing flies. Frequency distribution of ommatidia with different numbers of rhabdomeres three days after eclosion (hatching) in progeny of flies that express mutant huntingtin exon 1 (gmrQ120) and that had been crossed to either a control stock (w¹¹¹⁸) (white minus; have huntingtin transgene only) or to Rab5-EGFP flies (have huntingtin and Rab5 transgenes). P0.001, t-test; P0.001, Mann-Whitney U test. The rhabdomere frequency of Rab5 flies crossed to a control stock is also shown. (e) Odds ratio of GFP-expressing cells with Q74 aggregates in wild-type (Atg5^+/+) vs Atg5 knockout (Atg5^–/–) MEFs. ^***P<0.0001. (f) Odds ratio of Q74-expressing Atg5^–/– (Atg5 knockout) or Atg5^+/+ (wild-type) MEF cells with aggregates, after transient transfection with dominant-negative Rab5 (DN-Rab5), constitutive active Rab5 (CA-Rab5) or empty vector control and EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats (Q74) (3:1 ratio) for 48 hours. ^***P<0.0001. Odds ratios are given to compare pooled summary statistics across multiple independent experiments (see Materials and Methods). Control conditions are fixed at 1 in both cell lines to facilitate comparisons. (g) Quantification of GFP-expressing HeLa cells containing aggregates that had been transiently transfected with dominant-negative Rab5 (DN-Rab5) or empty vector (Cont) and huntingtin exon 1 with 74 polyglutamine repeats (Q74) (at a 3:1 ratio) for 48 hours and were either left untreated (–Rap) or treated with 0.2 µg/ml rapamycin (Rap) to induce autophagy. ^**P<0.001. Error bars in all graphs represent the s.e.m.

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