Figure 1.

Rab5 modulates mutant huntingtin toxicity/aggregation. a. Quantification of proportions of GFP-expressing cells with cell death in COS-7 cells transiently transfected with dominant-negative (DN), constitutive active (CA), wild-type (WT) Rab5 or empty vector control and EGFP-tagged huntingtin exon-1 with 74 polyglutamine repeats (Q74) (3:1 ratio) for 48h. *** - P < 0.0001, ** - P < 0.001, * - P < 0.05. b. Quantification of proportions of GFP-expressing cells with aggregates in COS-7 cells that were quantified for toxicity shown above. *** - P < 0.0001. c. Quantification of proportions of GFP-expressing cells with aggregates in HeLa cells transiently transfected with siRNA for Rab5a, Rab5b, Rab5c or all three siRNA simultaneously (Rab5abc) for 72h and further with EGFP-tagged huntingtin exon-1 with 74 polyglutamine repeats for the last 24h of the 72h siRNA transfection. *** - 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, 3 days after eclosion (hatching), in progeny of flies expressing mutant huntingtin exon-1 (gmrQ120) crossed to either a control stock (w¹¹¹⁸) (white minus) (have huntingtin transgene only) or to Rab5-EGFP flies (have huntingtin and Rab5 transgenes). P ≤ 0.001 t-test; P ≤ 0.001 also if tested by Mann-Whitney U test. The rhabdomere frequency of Rab5 flies crossed to a control stock is also shown. e. Proportion of GFP-expressing cells with Q74 aggregates in wild-type (Atg5+/+) vs Atg5 knockout (Atg5−/−) MEFs. *** - P < 0.0001. f. Quantification of proportions 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 48h. *** - P < 0.0001. Odds ratios are used to provide comparable pooled summary statistics across multiple independent experiments (see methods). Control conditions are fixed at 1 in both cell lines to facilitate comparisons. g. Quantification of proportions of GFP-expressing cells with aggregates in HeLa cells transiently transfected with dominant-negative Rab5 (DN-Rab5) or empty vector (Cont) and huntingtin exon-1 with 74 polyglutamine repeats (Q74) (3:1 ratio) for 48h and either left untreated (-Rap) or treated with 0.2μg/ml rapamycin (Rap) to induce autophagy. ** - P < 0.001. Error bars in all the above graphs represent S.E.M.