Figures & data
Figure 1. Effects of dj-1β mutation on mitochondrial physiology. (A) Levels of H2O2 production (nmol/min mg prot) shown as mean ± SEM. Between four to six replicate experiments per group. (B) Levels of superoxide production (AUF, arbitrary units of fluorescence) shown as mean ± SEM. Between four to six replicate experiments per group. (C) Mitochondrial oxygen consumption state 3 respiration (nmol O2/s mg prot) shown as mean ± SEM. Between three to six replicate experiments per group. (D) ATP content (µmol/mg protein) in fly homogenates shown as mean ± SEM, 10 replicate experiments per group. (E) Activity of individual flies recorded as average activity per hour (mean ± SEM) over a 48 h period is shown. Between 34–38 individual flies per group. (F) BNE gels of mitochondrial protein extracts from control and mutant flies. In gel, activity of complex I and IV is shown separately. p < 0.05 indicated by *. m, male; f, female.
Figure 2.dj-1β mutation increases oxidative damage in and outside mitochondria. (A) Levels of protein damage in fly homogenates mean ± SEM is shown. Between three to seven replicate experiments per group. (B) Levels of protein damage in fly mitochondria mean ± SEM is shown. Between three to six replicate experiments per group. GSA, glutamic semialdehyde; AASA, aminoadipic semialdehyde; CEL, carboxyethyl-lysine; CML, carboxymethyl-lysine; MDA-lys, malondialdehyde-lysine. Data are average ± SEM of at least three independent samples per group. p < 0.05 indicated by *. m, male; f, female.
Figure 3. Effect of dj-1β mutation on lifespan. (A) Survival of wild-type strains (DAH and OR) and dj-1β mutant flies (DJGE) at 25°C. n = 80 flies per experiment. Median (maximum) lifespan in days, males DAH 41 (51), males OR 65 (72), males DJGE 58 (60); females DAH 74 (79), females OR 81 (86), females DJGE 86 (86); DAH flies live significantly shorter than DJGE or OR (Log-Rank Test p < 0.001). (B) Survival of wild-type (DAH), dj-1β mutant flies (DJGE) and dj-1β mutant flies backcrossed for 11 generations into DAH (DJGE (DAH)) at 25°C. n = 80 flies. Median (maximum) lifespan in days, males DAH 35 (39), males DJGE 56 (74), males DJGE (DAH) 48 (63); females DAH 74 (79), females DJGE 79 (81), females DJGE(DAH) 77 (81); DAH flies live significantly shorter than DJGE or DJGE (DAH) (Log-Rank Test p < 0.001). (C) Survival of wild-type strains (DAH and OR) and mutant flies (DJGE) at 29°C. n = 200 flies per experiment. Median (maximum) lifespan in days, males DAH 38 (40), males OR 42 (47), males DJGE 36 (38); females DAH 46 (49), females OR 49 (56), females DJGE 46 (49); DJGE male flies live significantly shorter than DJGE or OR (Long Rank Test p < 0.001). No differences in lifespan between DJGE and DAH females. (D) Survival of wild-type (DAH) and dj-1β mutant flies (DJGE) at 25°C in high (HC) and low caloric- (LC) content diets. Male DAH 40 (54), male DJGE 65 (77), male DAH DR 40 (61), male DJGE DR 77 (89); female DAH 49 (68), female DJGE 77 (93), female DAH DR 74 (89), female DJGE DR 98 (108). DJGE flies live significantly longer than DAH in both diets (Log-Rank Test p < 0.001). m, male; f, female.
Figure 4. The dj-1β mutation activates pro-survival pathways in Drosophila melanogaster. (A) Q-PCR of Ilp’s 2, 3 and 5 from dj-1β mutant and control flies, normalized to Actin 88F. Data are presented as mean ± SEM (six replicate experiments). (B) Mitochondrial copy number assayed by Q-PCR of 16S rRNA gene from dj-1β mutant flies and control flies, normalized to levels of Rpl32. Data are presented as mean ± SEM (four to six replicate experiments). (C) Western blots analysis of complex I (NDUFS3 subunit) and loading control PDH (E1α subunit). Data are presented as mean ± SEM (four replicate experiments). (D) Sirtuin activity in homogenates of dj-1β mutant flies and control flies (five replicate experiments). p < 0.05 indicated by *. m, male; f, female. M, dj-1β mutant; C, control.
Figure 5. The dj-1β mutation accelerates development. (A) Number of eclosing flies per day, 10 different replicate experiments in control and dj-1β mutant flies. (B) Representative images of those taken daily during developmental time experiments. Stages from pupariation to eclosion are shown.
